Between 1874 and 1878, he studied medicine at the Kaiser-Wilhelm-Akademie in Berlin, an academy for military doctors, since his family could not afford the university. As a military doctor, he studied the action of iodoform. In 1888, he became an assistant at the institute of Robert Koch in Berlin.

In 1890 he published an article with Kitasato Shibasaburō reporting that they had developed “antitoxins” against both diphtheria and tetanus. They had injected diphtheria and tetanus toxins into guinea-pigs, goats and horses; when these animals developed immunity, they derived antitoxins (now known to consist of antibodies) from their serum. These antitoxins could protect against and cure the diseases in non-immunized animals. In 1892 he started the first human trials of the diphtheria antitoxin, but they were unsuccessful. Successful treatment started in 1894, after the production and quantification of antitoxin had been optimized. During 1894, Behring was also awarded the Cameron Prize for Therapeutics of the University of Edinburgh.

In 1895 he became Professor of Hygienics within the Faculty of Medicine at the University of Marburg, a position he would hold for the rest of his life. He and the pharmacologist Hans Horst Meyer had their laboratories in the same building, and Behring stimulated Meyer’s interest in the mode of action of tetanus toxin.

In December, 29th, 1896, Behring married the then twenty-year-old Else Spinola (1876-1936), who was a daughter of Bernhard Spinola [de], the director of the Charité hospital in Berlin, and a Jewish-born mother – Elise Spinola, born Bendix – who had converted to Christianity upon her marriage. They had six sons. They held their honeymoon at villa “Behring” on Capri 1897, where Behring owned a vacation home. In 1909–1911, the Russian writer Maxim Gorky lived at this villa.

Behring won the first Nobel Prize in Physiology or Medicine in 1901 for the development of serum therapies against diphtheria. He was elected a Foreign Honorary Member of the American Academy of Arts and Sciences in 1902.

Von Behring is believed to have cheated Paul Ehrlich out of recognition and financial reward in relation to collaborative research in diphtheria. The two men developed a diphtheria serum by repeatedly injecting the deadly toxin into a horse. The serum was used effectively during an epidemic in Germany. A chemical company preparing to undertake commercial production and marketing of the diphtheria serum offered a contract to both men, but von Behring maneuvered to claim all the considerable financial rewards for himself. To add insult to injury, only Behring received the first Nobel Prize in Medicine, in 1901, for his contributions. However, Ehrlich would go on to win the 1908 Nobel Prize in Medicine for his contribution to immunology.

In 1904 he founded the Behringwerke in Marburg, a company to produce antitoxins and vaccines.

At the International Tuberculosis Congress in 1905 he announced that he had discovered “a substance proceeding from the virus of tuberculosis.” This substance, which he designated “T C,” plays the important part in the immunizing action of his “bovivaccine”, which prevents bovine tuberculosis. He tried unsuccessfully to obtain a protective and therapeutic agents for humans.

Behring died at Marburg, Hessen-Nassau, on 31 March 1917. His name survived in the Dade Behring organisation (now part of the Siemens Heathineers), in CSL Behring, a manufacturer of plasma-derived biotherapies, in Novartis Behring and in the Emil von Behring Prize of the University of Marburg, the highest endowed medicine award in Germany.