Born and raised in Santa Ana, Orange County, California (about 30 miles southeast of Los Angeles) to Dick Pfeiffer and Donna Taverno, she is the second of four siblings: an older brother, Rick Pfeiffer, and two younger sisters, Dedee Pfeiffer (b. 1964, also an actress) and Lori Pfeiffer (b. 1965). Pfeiffer attended Fountain Valley High School and graduated in 1976. She briefly pursued a career as a court reporter at Golden West Community College before dropping out in pursuit of a career in acting. She entered the Miss Orange County beauty pageant in 1978, won and entered the statewide competition of Miss California. Though unsuccessful in claiming the title, the young Pfeiffer won herself an agent and roles in TV commercials and cameos before making her mark in Hollywood.

Pfeiffer's first major screen role was in the film sequel, Grease 2. However, it wasn't until 1983 when Pfeiffer starred with Al Pacino in Brian De Palma's gangster classic Scarface that caught the attention of Hollywood. Over the course of the 1980's and 1990's, Pfeiffer starred in a string of box office and critical hits as Ladyhawke, The Witches of Eastwick, Married to the Mob, Tequila Sunrise, The Russia House, Frankie and Johnny, Batman Returns, The Age of Innocence, Dangerous Minds, and One Fine Day. She was honored with the British Academy Award and also received her first Oscar nomination in 1988 as Best Supporting Actress in Stephen Frears's Dangerous Liaisons. Pfeiffer was honored with her second Best Actress Academy Award nomination in 1992's Love Field, which also won Pfeiffer the Silver Bear at the Berlin International Film Festival in 1993. However, it is Pfeiffer's role as chanteuse Susie Diamond in 1989's The Fabulous Baker Boys that continues to be synonymous as being the highlight of her career. Pfeiffer's performance garnered another Academy Award nomination as Best Actress, as well as honors from the Hollywood Foreign Press, New York Film Critics, Los Angeles Film Critics, as well as the National Society of Film Critics and Board of Review. Critics compared Pfeiffer's performance, much known for her scorching rendition of "Makin Whoopee" atop of a piano, as being rightfully in the ranks of Marilyn Monroe, Rita Hayworth, and the young Katharine Hepburn. In her rave review of the film, the late Pauline Kael recalled Pfeiffer's performance as having "the grinning infectiousness of Carole Lombard and the radiance of the very young Lauren Bacall."

In 1995, Pfeiffer was given the Hasty Pudding Woman of the Year award by Harvard University for her contribution to the performing arts. The award, bestowed annually by the Hasty Pudding Theatricals society, is given to Female performers who they deem to have made a "lasting and impressive contribution to the world of entertainment." The award, has placed Pfeiffer in the ranks of Katharine Hepburn, Elizabeth Taylor, and Meryl Streep, among others.

Since then, Pfeiffer has continued to maintain her status as one of the film world's reigning screen goddesses, despite working much less in recent years. In 2000, Pfeiffer starred with Harrison Ford in one of the biggest box office hits of that year, in Robert Zemeckis's thriller What Lies Beneath. In 2001, Pfeiffer starred opposite Sean Penn in I Am Sam and in 2002 alongside Renee Zellweger and Robin Wright Penn in White Oleander, which earned Pfeiffer the Screen Actors Guild Award nomination as Best Supporting Actress. Currently, Pfeiffer is busy with upcoming projects that has her teaming up with Clueless director Amy Heckerling in the romantic comedy I Could Never Be Your Woman (opposite Paul Rudd, released Summer 2006) as well as the Sci-Fi fantasy epic opposite Robert DeNiro, Claire Danes, and Sienna Miller in Stardust, due to be released in 2007. It is also rumored that she will co-star in the film adaptation of the hit Broadway musical Hairspray as Velma Van Tussle.

In 1981, Pfeiffer married Thirtysomething actor Peter Horton, but later divorced in 1988 at the height of Pfeiffer's career. She has been linked romantically with actors Val Kilmer, John Malkovich, Michael Keaton, and Fisher Stevens. In 1993, Pfeiffer married writer-producer David E. Kelley (The Practice, L.A. Law, Ally McBeal, Boston Legal). That same year Pfeiffer adopted a girl, Claudia Rose Kelley, and a year later welcomed their son, Jack Henry Kelley.

Currently, Pfeiffer and Kelley divide their time between homes in Los Angeles and Northern California.