Fleming was born in Indiana, Pennsylvania, on February 14, 1959. Her parents were high school vocal music teachers in Rochester, New York, where she mostly grew up. In 1981, she graduated from the State University of New York at Potsdam (where she sang in a jazz trio at a bar) with a degree in music education and continued her musical studies at the Eastman School of Music, which she credits with giving her a strong academic and theoretical background. From 1983 to 1987, she was enrolled in the American Opera Center at Juilliard, where she met Beverley Johnston, the voice teacher with whom she would continue to study throughout her career. Fleming also recalls with admiration the year she spent studying Lieder with Arleen Augér, on a Fulbright Scholarship. In 1988, she won the Metropolitan Opera National Council Auditions and the George London Prize (in the same week), and the Eleanor McCollum Competition in Houston.
Fleming sang the Countess in Mozart's Le nozze di Figaro at the Houston Grand Opera in 1988, made her New York City Opera debut in 1989 as Mimi in La bohème, and her Covent Garden debut as Glauce in Cherubini's Medea later that year. In 1991, she made her acclaimed Met debut, stepping in for an indisposed Felicity Lott as the Countess in The Marriage of Figaro. The Countess was her debut role in San Francisco (1991), Vienna State Opera (1993), and Glyndebourne (1994). In 1993, she made her debut at La Scala as Donna Elvira, and she sang Eva at the 1996 Bayreuth Festival. Since that time, she has continued performances at the world's leading opera houses and concert halls and has continued to expand her repertoire. Among the roles for which she has won acclaim are Handel's Alcina and Rodelinda; Rossini's Armida, Violetta, Manon, Thaïs, Tatyana, and Rusalka; and numerous roles in Strauss operas, including the Marschallin, Daphne, Arabella, and the Countess in Capriccio. She created the role of Rosina in Corigliano's The Ghosts of Versailles in 1991, Madame Tourvel in Conrad Susa's Dangerous Liaisons in 1994, and Blanche DuBois in André Previn's A Streetcar Named Desire in 1998.
She has garnered praise for her many recordings, both on CD and DVD, has been nominated for 12 Grammy Awards and has won four, in 1996, 1999, 2010, and 2013. In addition to her work in the classical repertoire, Fleming has recorded contemporary pop songs, jazz, and film soundtracks. She has hosted a number of television and radio broadcasts, including The Metropolitan Opera's Live in HD series, and Live from Lincoln Center for PBS. Her honors include Sweden’s Polar Prize (2008), designation as Chevalier de la Légion d’Honneur from the French government (2005), Honorary Membership in the Royal Academy of Music (2003), and a 2003 Honorary Doctorate from the Juilliard School. In 2012 she received the U.S. National Medal of the Arts from President Barack Obama.
Fleming's activities in mid-career have continued to show energy in traditional channels, but she has also, while generally avoiding "crossover" releases, made use of her background in vernacular music. She has appeared, among many other roles at top houses, as Desdemona in Verdi's Otello in at London's Covent Garden in 2005 and at the Metropolitan Opera in New York in 2008-2009. Her recordings have included a 2013 production of Richard Strauss' Ariadne auf Naxos from Baden-Baden, Germany, released by Decca on DVD in 2013. Her recording schedule has intensified; since 2003 she has released at least 15 CDs, covering everything from traditional repertory to jazz (Haunted Heart, 2005, and Love Sublime, 2006) and indie rock (Dark Hope, 2010, and the group of Björk songs included on Distant Light, 2017). Fleming has avoided traditional operatic stylings in her pop and jazz releases. She is the author of a memoir, The Inner Voice: The Making of a Singer.