Hofmann studied under Justus von Liebig at the University of Giessen and received his doctorate in 1841. In 1845 he became the first director of the new Royal College of Chemistry, in London. He moved to Bonn in 1864 but in 1865 became chemistry professor and laboratory director at the University of Berlin, continuing there as a teacher and researcher until his death.

His work covered a wide range of organic chemistry. His first research, on coal tar, led to the development of practical methods for obtaining benzene and toluene and converting them into nitro compounds and amines. In other work he prepared the three ethylamines and tetraethylammonium compounds and established their structural relationship to ammonia. He discovered formaldehyde, hydrazobenzene, the isonitriles, and, with Auguste Cahours, allyl alcohol. The Hofmann reaction was named after his method of converting an amide into an amine. He also developed a method for determining the molecular weights of liquids from vapour densities. Almost 1,000 scientific papers came from his laboratory, and nearly 300 of these represented his own work. He was a cofounder of the German Chemical Society (1867) and served 14 terms as its president during the years 1868–92.