Miescher came from a distinguished scientific family from Basel in Switzerland: both his father, also called Johann Friedrich, and his uncle, Wilhelm His, held the chair of anatomy at the University of Basel. Miescher himself studied medicine at Basel but, feeling that his partial deafness (produced by a severe attack of typhus) would be a drawback for a physician, turned to physiological chemistry. He consequently spent the period from 1868 to 1870 learning organic chemistry under Felix Hoppe-Seyler at Tübingen and physiology at Leipzig in the laboratory of Carl Ludwig. In 1871 he was appointed professor of physiology at Basel.

It was while working on pus cells at Tübingen in 1869 that Miescher made his fundamental discovery. It was thought that such cells were made largely of protein, but Miescher noted the presence of something that “cannot belong among any of the protein substances known hitherto.” In fact he was able to show that it was not protein at all, being unaffected by the protein-digesting enzyme pepsin. He also showed that the new substance was derived from the nucleus of the cell alone and consequently named it ‘nuclein’. Miescher was soon able to show that nuclein could be obtained from many other cells and was unusual in containing phosphorus in addition to the usual ingredients of organic molecules – carbon, oxygen, nitrogen, and hydrogen. It was not until 1871 that Miescher's paper, delayed by Hoppe-Seyler (who wanted to confirm the results), was published. In it he announced the presence of a nonprotein phosphorus- containing molecule in the nuclei of a large number of cells.

Just what precise role the molecule played in the cell was not revealed until the structure of nucleic acid, as it was renamed by Richard Altmann in 1889, was announced by James Watson and Francis Crick in 1953. Miescher continued to work on the nuclein extracted from the sperm of the Rhine salmon for the rest of his short life. He spent much time puzzling on the chemistry of fertilization, even speculating in 1874 that “if one wants to assume that a single substance…is the specific cause of fertilization then one should undoubtedly first of all think of nuclein.” Unfortunately Miescher failed to follow up his suggestion, preferring to explore physical models of fertilization. However, his work on nuclein was eagerly taken up by other organic chemists, and by 1893 Albrecht Kossel had succeeded in recognizing four nucleic acid bases.