Dushku was born in Watertown, Massachusetts, the daughter of Philip Dushku, an administrator and teacher in the Boston Public Schools, and Judith, a political science professor. Dushku's father is Albanian and her mother is of Danish and English descent. Dushku attended Beaver Country Day School in Chestnut Hill, and graduated from Watertown High School. She was raised in The Church of Jesus Christ of Latter-day Saints, the faith of her mother, though she is not practicing. She has three older brothers: Aaron, Benjamin and Nathaniel. Her parents divorced when she was an infant.
Dushku came to the attention of casting agents when she was 10. She was chosen in a five month search for the lead role of Alice in the film That Night. In 1993, Dushku landed a role as Pearl alongside Robert De Niro and Leonardo DiCaprio in This Boy's Life, a role that she said opened a lot of doors. The following year, she played the teenage daughter of Arnold Schwarzenegger and Jamie Lee Curtis in True Lies. She had parts as Paul Reiser's daughter in Bye Bye Love, as Cindy Johnson in Race the Sun, and roles in a television movie and a short film.
Dushku took time off from acting to finish her junior and senior years of high school. She was accepted to the George Washington University in Washington, D.C. and Suffolk University in Boston, where her mother serves as professor of government and previously served as dean of the campus in Dakar, Senegal.
After completing high school, Dushku returned to acting with the role of Faith on Buffy the Vampire Slayer, a Slayer much more troubled than the main character Buffy Summers. Though initially planned as a five episode role, the character became so popular that she stayed on for the whole third season and returned for a two-part appearance in season four, after which the remainder of her original story arc was played out as part of the first season of the Buffy spin-off series Angel.
In 2000, Dushku starred in the hit cheerleader comedy Bring It On. She followed that up with Soul Survivors, reuniting her with Race The Sun co-star Casey Affleck. In 2001, she appeared in The New Guy with DJ Qualls and City by the Sea with Robert De Niro and James Franco. The same year, Kevin Smith invited Dushku to be a part of Jay and Silent Bob Strike Back. In 2003, Dushku starred the horror film Wrong Turn, and The Kiss, an independent comedy-drama. Starting that same year, she starred in a new Fox TV series, Tru Calling, where she played the main character, medical student Tru Davies. Dushku turned down a role in a spin-off of Buffy The Vampire Slayer which would have been about Faith.
Dushku starred in an Off-Broadway production entitled Dog Sees God from December 2005, playing "Van's sister", a character paralleled with Lucy Van Pelt from the Peanuts comic strip on which the play production is based. She quit in February 2006 along with other members of the cast amidst rumors of abuse from the producer which were later dismissed. She played the lead character on Nurses, a hospital comedy/drama for Fox. She also appeared in the Simple Plan music video, "I'm Just a Kid", as the band's love interest, as well as Nickelback's video for "Rockstar".
On October 1, 2005, she announced at Wizard World Boston that shooting had begun for Nobel Son in which she would star with Alan Rickman, Danny DeVito, Bill Pullman, and Peter Boyle. The movie was released at the 2007 Tribeca Film Festival. Another project is On Broadway, an independent movie filmed in her native Boston. Variety announced on August 2, 2006 that Dushku would co-star with Macaulay Culkin in Sex and Breakfast, a dark comedy written and directed by Miles Brandman. She starred in Open Graves, a 2008 horror-thriller about a satanic game co-starring Mike Vogel. She played the main character in The Thacker Case and The Alphabet Killer, both thrillers based on real-life events, one of them directed by Rob Schmidt with whom she had worked on Wrong Turn.
On August 26, 2007, Dushku signed a development deal with Fox Broadcasting and 20th Century Fox. Under the pact, the network and the studio would develop projects tailor-made for the actress. Consequently, it was announced on October 31 that Dushku had lured Joss Whedon, of Buffy The Vampire Slayer, back to TV, as they agreed to create a show called Dollhouse. Dushku produced the show and played the main character, 'Echo', which aired on Fox during the 2008-09 TV Season.
Dushku was the voice actor for contract killer "Rubi Malone" in the action video game Wet (2009). Dushku secured exclusive rights to make "The Perfect Moment", a film based on the life of photographer Robert Mapplethorpe and enlisted the help of Ondi Timoner. Dushku provided her voice for Noah's Ark: The New Beginning and appeared in the film Locked In which was released in 2010. Dushku guest-starred in CBS' comedy The Big Bang Theory in the fall of 2010.
In 2011, Dushku featured alongside Jayson Floyd in "One Shot", a short action clip on YouTube directed by and starring Freddie Wong, which was released on May 13, 2011. Dushku will have a lead role in an online animated "motion comic" series, titled Torchwood: Web of Lies, based on Starz' upcoming Torchwood: Miracle Day. In August 2011, Dushku visited Albania to film a documentary on her father's country of origin with a crew from the Travel Channel and Lonely Planet.
Dushku resides in Laurel Canyon, Los Angeles, California. Dushku is the CEO of her production company, Boston Diva Productions. In 2006, she visited her father's family in Albania after an invitation from the Prime Minister of Albania, Sali Berisha. She visited Kosovo and got an Albanian Eagle tattoo on the back of her neck. Dushku began dating former Los Angeles Lakers basketball player Rick Fox in October 2009, and in August 2010 the couple confirmed that they are living together.
While on her second visit to Albania in 2011, she applied for Albanian citizenship and obtained the Albanian passport and ID Card. She became an honorary citizen of Tirana, and was given the honorary title of Tirana Ambassador of Culture and Tourism in the World by Tirana mayor Lulzim Basha. Additionally, she was given honorary citizen status in her father's home town of Korce, Albania. She serves on the board of directors of the THARCE-Gulu organization (The Trauma Healing and Reflection Center in Gulu), an organization dedicated to helping the survivors of war (including former child soldiers) in Northern Uganda).