Educated at the University of Washington and Yale University, where she earned her Ph.D. in 1968, Middlebrook wrote her doctoral dissertation on Walt Whitman and Wallace Stevens. She claimed the dissertation wasn't very good, and that "you can’t find my book in the library, because it isn't in the Whitman place, it isn't in the Stevens place, it's in some American literature place."

Middlebrook began her teaching career at Stanford as an assistant professor in 1966 and gradually worked her way up to university professor and associate dean positions. She won a number of fellowships, grants, and awards along the way. She resigned from Stanford in 2004 to concentrate fully on her writing. By this time, she was already a professor emerita.

After the book on Stevens and Whitman, Middlebrook wrote or edited three books on poetry: Worlds Into Words: Understanding Modern Poems (1980), Coming to Light: American Women Poets in the 20th Century (1985), and Selected Poems of Anne Sexton (1988). She also wrote her own book of poetry, Gin Considered as a Demon (1983). The edition of Anne Sexton's poetry helped lead to what can be described as Middlebrook's big break: her book Anne Sexton, A Biography published in 1991.

The Sexton biography became a New York Times bestseller and a finalist for the National Book Award. An intriguing blend of traditional biography, psychiatric study, and literary criticism, the book was written in an infectious, non-pedantic style that attracted a wide range of readers.

Middlebrook then took an interesting detour from her study of poetry and poets in her next book, Suits Me: The Double Life of Billy Tipton (1998). The story of a jazz musician who was born a woman but lived for over fifty years as a man, the book also sold well and showed that Middlebrook could range outside the traditional purview of the English Department.

The Sexton biography might have led inevitably to Middlebrook's book about Anne Sexton's friend and fellow-suicide, Sylvia Plath. Published as Her Husband: Ted Hughes & Sylvia Plath, a Marriage in 2003, the biography steered a sensible and convincing course between partisan views of the two poets. Publishers Weekly called it the "gold standard" of the many books published about the couple, and it became a Los Angeles Times bestseller.

Besides her books, Middlebrook published many articles on Sexton, Plath, Hughes, and other writers, such as Robert Lowell and Philip Larkin. She also reviewed a wide variety of books on subjects ranging from Helen Keller to the development of modern clothing. She said that her planned book on Ovid was an attempt to get inside a subject who "exists only in his texts."

Born as Diane Wood, hailing from Pocatello, Idaho, Middlebrook spent the last 28 years of her life with her third husband, Carl Djerassi, a Viennese-born American scientist who helped invent the first contraceptive pill. Middlebrook's experience with her two divorces was something that, in her own words, "rips your soul out of your body".

She and her husband spent the winters in San Francisco and the summers in London. According to her family, Middlebrook died of cancer