O'Keefe made her big screen debut in 1998 in Halloween H20: 20 Years Later and later starred in such films as The Crow: Salvation, Whatever It Takes and Devil In The Flesh 2. In 1999, she co-starred in the teen film She's All That (1999) as Taylor Vaughan alongside Freddie Prinze Jr. and Rachael Leigh Cook. When asked if it was hard to play her character, a high school prima donna, she replied, “Everybody knew a Taylor Vaughan in high school. Working in show Business, you meet girls like that every day.”
O'Keefe kept on filming both movies and Nash Bridges until the series ended in 2001. Later films have included Out for Blood, in which she played a vampire named Layla Simmons and Venice Underground. On the small screen, she has appeared in various shows including Dharma & Greg, Boston Legal, Two and a Half Men, Charmed, The Evidence, The Big Bang Theory, and Tru Calling.
In 2005 she played Isabella on an episode of Two and a Half Men.
In 2007, O'Keefe was cast in a starring role on the series Prison Break; she was a part of the show's cast through its third and fourth seasons. O'Keefe has said that she "truly loved" playing the part of her Prison Break character Gretchen Morgan, because it "challenged" her, and because Gretchen was the "polar opposite of her" and "was badass".
In 2009 she appeared in the stand-alone add-on of the video game Command & Conquer: Red Alert 3: Uprising.
On June 22, 2011, O'Keefe started her own clothing line, Queen George Clothing.
In 2012, O'Keefe launched her own jewelry line, Q.
In June 2013, O'Keefe did a photoshoot for NOH8 campaign.
In 2014 O'Keefe was cast in a recurring role in VH1's TV series Hit the Floor, and appeared in the film Merry ExMas. The same year, she was also cast in the recurring role of Jo in the sixth season of The CW series The Vampire Diaries.
O'Keefe started her career as a model at the age of 8 years old, and she did some modeling work for a Jeans company. Midway through her junior year of high school, O'Keefe left school to star on the soap opera Another World, playing Marguerite “Maggie” Cory. She then got a role on Nash Bridges playing Cassidy, the daughter of Don Johnson’s title character. She and her mother moved to Hollywood so O'Keefe completed her schooling by mail.