Biffen was born in Cheltenham and, after graduating in natural sciences from Cambridge in 1896, joined a team investigating rubber production in Mexico, the West Indies, and Brazil. On his return he was appointed lecturer in botany at Cambridge and patented a method for handling rubber latex.

Biffen was inclined more toward applied than pure botany and joined the Cambridge School of Agriculture shortly after its foundation in 1899. He began conducting cereal trials in order to select improved types, and when Gregor Mendel's laws of inheritance were rediscovered in 1900, he realized immediately that they could be applied to improving plant-breeding methods. Biffen speculated that physiological as well as morphological traits would prove to be inherited in Mendelian ratios, and in 1905 demonstrated that this was true for resistance to yellow rust, a fungal disease of wheat.

Little Joss and Yeoman, two wheat varieties bred by Biffen, were unequaled for many years. In 1912 Biffen became director of the Plant Breeding Institute at Trumpington, a newly formed research center established by the government to promote Biffen's work and the application of scientific principles to plant breeding. Biffen was also professor of agricultural botany at the university from 1908 to 1931 and was instrumental in setting up the National Institute of Agricultural Botany at Cambridge. He was knighted for his services to agriculture in 1925.