Julia O'Hara Stiles was born on March 28, 1981 in New York City, the outgoing daughter of a Greenwich Village artist mother, Judith Newcomb Stiles, and an elementary school teacher father, John O'Hara. She is the eldest of three children, and has Irish, English, and German ancestry. Encouraged to take modern dance lessons at an early age, she was introduced to Shakespeare and theater. At age 11, she made her debut as a child actress with the experimental off-Broadway La MaMa Theatre. Her passion grew and by the next year was performing professionally and working in commercials. A bright, precocious student, she was seriously considered for the child vampire role of Claudia in Interview with the Vampire: The Vampire Chronicles (1994) but lost out in the end to Kirsten Dunst. She continued training at New York's Professional Children's School and, at 15, made her cinematic bow with I Love You, I Love You Not (1996) with Claire Danes. Next featured as the daughter of Harrison Ford in The Devil's Own (1997), her breakout role came on TV with the hard-hitting mini-movie Before Women Had Wings (1997), which was produced by Oprah Winfrey and dealt with child abuse. Her wish to play Shakespeare was granted threefold with her participation in 10 Things I Hate About You (1999), which was based on the Bard's "The Taming of The Shrew" and won her the Chicago Film Critics Award as the volatile teen Kat; an updated version of Hamlet (2000) which paired her Ophelia with Ethan Hawke; and another updated version of "Othello" entitled O (2001), which had her high school character of Desi (Desdemona) involved in a mixed romantic relationship with African-American Mekhi Phifer. The violent-edged film was made in 1998 but not released until three years later due to the tragic Colorado student shootings at Columbine High School. In addition, Julia later portrayed Viola off-Broadway in a Shakespeare-in-the-Park production of "Twelfth Night" in 2002.

She temporarily interrupted her career after deciding to enroll at Columbia University in 2000, where she earned a degree in English. Stiles has also returned to the stage, appearing in David Mamet's "Oleanna" in London, a role she would reprise on Broadway in 2009, opposite Bill Pullman and directed by Doug Hughes.

Moving into mainstream roles by the millennium, she took on a role that would carry her through the decade, co-starring with Matt Damon in popular Bourne series: The Bourne Identity (2002), The Bourne Supremacy (2004), and The Bourne Ultimatum (2007). During the time between Bourne films, she appeared in ensemble dramas, appearing opposite Julia Roberts in Mona Lisa Smile (2003), won offbeat notice in the title role of Carolina (2003) with Shirley MacLaine and Randy Quaid. She also returned to her indie film roots, with films such as Between Us (2012) and the comedy farce, It's a Disaster (2012).

In 2010, she switched gears and headed to series television, joining the cast of Dexter (2006) for the show's fifth season in the role of Lumen Pierce, a performance that earned her nominations for both the Golden Globe and the Emmy for Best Supporting Actress. In 2012, she followed up with another series, Blue (2012), which airs on YouTube, while preparing for cinematic roles in Silver Linings Playbook (2012) and The Bell Jar.