Behring was born in Hansdorf in Germany. He graduated in medicine at Berlin University and entered the Army Medical Corps before becoming (in 1888) a lecturer in the Army Medical College, Berlin. In 1889 he moved to Robert Koch's Institute of Hygiene and transferred to the Institute of Infectious Diseases in 1891, when Koch was appointed its chief.

In 1890, working with Shibasaburo Kitasato, Behring showed that injections of blood serum from an animal suffering from tetanus could confer immunity to the disease in other animals. Behring found that the same was true for diphtheria and this led to the development of a diphtheria antitoxin for human patients, in collaboration with Paul Ehrlich. This treatment was first used in 1891 and subsequently caused a dramatic fall in mortality due to diphtheria.

Behring's success brought him many prizes, including the first Nobel Prize in physiology or medicine, awarded in 1901. He was appointed professor of hygiene at Halle University in 1894 and one year later moved to a similar post at Marburg. In 1913 he introduced toxin–antitoxin mixtures to immunize against diphtheria, a refinement of the immunization technique already in use. He also devised a vaccine for the immunization of calves against tuberculosis.