Tomei was born and raised in Brooklyn to an Italian American family. While growing up, she was captivated by the Broadway shows her theater-loving parents took her to, and became drawn to acting as a career for herself. She attended Boston University for a year, then transferred to New York University in 1983 after landing a role in the soap opera As the World Turns (she would soon drop out of college as her career kicked into gear).

She followed up As the World Turns in 1987 with a role on the sitcom A Different World. Her breakthrough performance came in My Cousin Vinny (1992) for which she won an Oscar for Best Supporting Actress. She received a second Oscar nomination for Best Supporting Actress for In the Bedroom (2001).

She has also done substantial work in the theater world, including lead roles on Broadway in Wait Until Dark (1998) and Salomé (2003), and many Off-Broadway plays.

In 1996, she made a guest appearance on the sitcom Seinfeld, playing herself.

She also makes an appearance on The Simpsons as a movie star who falls in love with Ned Flanders.

In 2006, she had a recurring role on the TV series Rescue Me, playing Johnny's ex-wife Angie.

There is an urban legend that Tomei's Oscar for My Cousin Vinny should have gone to one of the four other nominees in 1993 (Miranda Richardson, Joan Plowright, Judy Davis or Vanessa Redgrave). The rumor claims

During the 'Oscar' ceremony, Jack Palance announced Tomei as best supporting actress winner, while actually there was another name on his note. The mistake was considered too embarrassing to correct.

This rumor has been debunked by Snopes.