Pompeo was born in Everett, Massachusetts, then lived on New York's Upper West Side. Her father was born in Gesualdo, a village in province of Avellino (Italy). She eventually moved to Miami, where she worked as a cocktail waitress and met New York fashion photographer Andrew Rosenthal, with whom she eventually returned to New York. In 1996 she was bartending at the SoHo Bar & Grill when an agent approached her about appearing in commercials. She subsequently appeared in commercials for Citibank and L'Oreal. Minor roles on shows like Strangers with Candy and a handful of films followed.

Determined to succeed in acting, Pompeo moved to Los Angeles in 2001. A turning point came when she was tapped by director Brad Silberling in 2002 for a role in his film Moonlight Mile. She was also in the film Daredevil playing Matt Murdock's (Ben Affleck) secretary Karen Page, but most of her scenes were deleted from the theatrical version of the film. She starred in Old School as Nicole and Catch Me If You Can as Marci. Guest roles include two episodes of Law & Order and an episode of Friends ("The One Where the Stripper Cries").

However, Pompeo landed her first major role in the ABC medical drama Grey's Anatomy, as the title character, Meredith Grey, an intern and lead character at a prestigious hospital.