Biel began her career as a vocalist appearing in musical productions until she was cast as Mary Camden in the family drama series 7th Heaven (1996–2006), in which she achieved recognition.

Biel has since starred in such films as The Rules of Attraction (2002), Blade: Trinity (2004), Stealth (2005), The Illusionist (2006), I Now Pronounce You Chuck & Larry (2007), Valentine's Day (2010), The A-Team (2010), New Year's Eve (2011), Total Recall (2012), and Hitchcock (2012).

[12][13] Biel's family moved frequently during her childhood, living in Texas, Connecticut, and Woodstock, Illinois, before finally settling in Boulder, Colorado.

At age 14, after auditioning for several television pilots, Biel was cast as Mary Camden, the oldest daughter and second-oldest child in the family drama 7th Heaven.

[18] Biel landed her first feature film role as Peter Fonda's granddaughter in the critically acclaimed drama Ulee's Gold, released in 1997.

James Berardinelli of Reelviews called her the "film's real acting revelation",[27] while Todd McCarthy of Variety wrote, "Handily employing a refined English accent where the others lay on a light Austrian veneer, Biel is entirely stunning enough to fight to the death over.

In late 2007, Biel signed on to play a stripper in Powder Blue, alongside Forest Whitaker (who also produced the film), Ray Liotta and Patrick Swayze.

[31][32] The Hollywood Reporter described her performance as "an irresistible force of nature — a kind, witty, supremely intelligent and beautiful woman who ... is capable of rejoinders that thoroughly undercut her opponent's withering criticism.

Biel performed the role of Sarah Brown with the Los Angeles Philharmonic in a fully staged concert production of Guys and Dolls during the 2009 season at the Hollywood Bowl.

[9] She subsequently landed a part in Lincoln Center Theater's two-week-long workshop of the musical version of the Pedro Almodóvar film Women on the Verge of a Nervous Breakdown, along with Salma Hayek.

[41] In 2015, Biel starred in the independent drama Bleeding Heart, in which she plays a yoga instructor named May who meets her biological sister Shiva (Zosia Mamet), a sex worker, for the first time.

Richard Lawson of Vanity Fair commented: "Though she's not given too much to work with in terms of character, Biel plays May with appealing nuance, creating a low-key, bliss-based Angeleno, all light and airy and gentle and poised, who discovers within herself an untapped hardness and anger and strength... when Biel has to conjure up more profound emotions, she proves adept, and surprisingly subtle.

"[44] In 2016, Biel co-starred with Patrick Wilson (with whom she had worked on The A-Team) in the thriller A Kind of Murder, based on the novel The Blunderer by Patricia Highsmith.

[50] Biel is set to lead the upcoming sci-fi thriller Ursa Major directed by brothers Jonathan and Josh Baker.

[58][59][60] On July 18, 2006, Biel participated in a charity auction to raise medical funds for teen Molly Bloom, who was injured in a limousine accident.

[63] In 2010, Biel climbed to the summit of Mount Kilimanjaro with members of the United Nations Foundation to raise awareness of the global water crisis.

[65] Biel teamed with nonprofit health care organization WomanCare Global to develop content that will provide girls with sex education.

[66] In 2021, Biel teamed up with Jeremy Adams, a natural products entrepreneur, and co-founder Greg Willsey, to launch Kinderfarms, a health and wellness brand for families.