Franklin was a Londoner by birth. After graduating from Cambridge University, she joined the staff of the British Coal Utilisation Research Association in 1942, moving in 1947 to the Laboratoire Centrale des Services Chimique de L'Etat in Paris. She returned to England in 1950 and held research appointments at London University, initially at King's College from 1951 to 1953 and thereafter at Birkbeck College until her untimely death from cancer at the age of 37.

Franklin played a major part in the discovery of the structure of DNA by James Watson and Francis Crick. With the unflattering and distorted picture presented by Watson in his The Double Helix (1968) her role in this has become somewhat controversial. At King's, she had been recruited to work on biological molecules and her director, John Randall, had specifically instructed her to work on the structure of DNA. When she later learned that Maurice Wilkins, a colleague at King's, also intended to work on DNA, she felt unable to cooperate with him. Nor did she feel much respect for the early attempts of Watson and Crick in Cambridge to establish the structure.

The causes of friction were various ranging from simple personality clashes to, it has been said, male hostility to the invasion of their private club by a woman. Despite this unsatisfactory background Franklin did obtain results without which the structure established by Watson and Crick would have been at the least delayed. The most important of these was her x-ray photograph of hydrated DNA, the so-called B form, the most revealing such photograph then available. Watson first saw it in 1952 at a seminar given by Franklin, and recognized that it clearly indicated a helix. Franklin also appreciated, unlike Watson and Crick, that in the DNA molecule the phosphate groups lie on the outside rather than inside the helix.

Despite such insights it was Watson and Crick who first realized that DNA has a double helix. By March 1953 Franklin had overcome her earlier opposition to helical structures and was in fact producing a draft paper on 17 March 1953, in which she proposed a double-chain helical structure for DNA. It did not, however, contain the crucial idea of base pairing, nor did she realize that the two chains must run in opposite directions. She first heard of the Watson–Crick model on the following day.