Flexner was born in Louisville, Kentucky on November 13, 1866. He was the sixth of nine children born to German Jewish immigrants, Ester and Moritz Flexner. He was the first in his family to complete high school and go on to college. In 1886, at age 19, Flexner completed a Bachelor of Arts in classics at Johns Hopkins University, where he studied for only two years. In 1905, he pursued graduate studies in psychology at Harvard University, and at the University of Berlin. He did not, however, complete work on an advanced degree at either institution.

In 1896, Flexner married a former student of his school, Anne Laziere Crawford. She was a Teacher who soon became a successful Playwright and children's author. The success of her play Mrs. Wiggs of the Cabbage Patch (based on the 1901 novel) funded Flexner's studies at Harvard and his year abroad at European universities. The couple had two daughters Jean and Eleanor. Jean went on to become one of the original employees of the United States Division of Labor Standards. Eleanor Flexner became an independent scholar and pioneer of women's studies.

The success of Abraham Flexner's experimental schooling allowed him to help Finance Simon Flexner's medical education at Johns Hopkins School of Medicine. He proceeded to become a pathologist, bacteriologist and a medical researcher employed by the Rockefeller Institute for Medical Research from 1901 to 1935.

His book attracted the attention of Henry Pritchett, President of the Carnegie Foundation, who was looking for someone to lead a series of studies of professional education. The book consistently cited Pritchett in discussions of views on educational reform, and the two soon arranged to meet through the then-president of Johns Hopkins University, Ira Remsen. Although Flexner had never set foot inside a medical school, he was Pritchett's first choice to lead a study of American medical education, and soon joined the research staff at the Carnegie Foundation in 1908. Although not a physician himself, Flexner was selected by Pritchett for his writing ability and his disdain for traditional education.

In 1910, Flexner published the Flexner Report, which examined the state of American medical education and led to far-reaching reform in the training of doctors. The Flexner Report led to the closure of most rural medical schools and all but two African-American medical colleges in the United States, given his adherence to germ theory, in which he argued that if not properly trained and treated, African-Americans and the poor posed a health threat to middle/upper class whites. His position was:

Between 1912 and 1925, Flexner served on the Rockefeller Foundation's General Education Board, and after 1917 was its secretary. With the help of the board, he founded another experimental school, the Lincoln School, which opened in 1917, in cooperation with the faculty at Teachers College of Columbia University.

In his 1930 Universities: American, English, German, Flexner returned to his earlier interest in the direction and purpose of the American university, attacking distractions from serious learning, such as intercollegiate athletics, student government, and other student activities.

During his time there, Flexner helped bring over many European Scientists who would likely have suffered persecution by the rising Nazi government. This included Albert Einstein, who arrived at the Institute in 1933 under Flexner's directorship.

Flexner died in Falls Church, Virginia, in 1959 at 92 years of age. He was buried in Cave Hill Cemetery in Louisville, Kentucky.

Flexner soon conducted a related study of medical education in Europe. According to Bonner (2002), Flexner's work came to be "nearly as well known in Europe as in America." With funding from the Rockefeller Foundation, Flexner "...exerted a decisive influence on the course of medical training and left an enduring mark on some of the nation's most renowned schools of Medicine." Bonner worried that "the imposition of rigid standards by accrediting groups was making the medical curriculum a monstrosity," with medical students moving through it with "little time to stop, read, work or think." Bonner (2002) calls Flexner "the severest critic and the best friend American Medicine ever had."