Burbank was brought up on a farm in Lancaster, Massachusetts, and received only an elementary education. He began breeding plants in 1870, when he bought a seven-hectare plot of land. After about a year he had developed the Burbank potato, which was introduced to Ireland to help combat the blight epidemics. By selling the rights to this potato he made $150, which he used to travel to California, where three of his brothers had already settled.

Burbank established a nursery and experimental farm in Santa Rosa, where the climate was especially conducive to fruit and flower breeding – his occupation for the next 50 years. He worked by making multiple crosses between native and introduced strains, using his remarkable skill to select commercially promising types. These were then grafted onto mature plants to hasten development, so that their value could be rapidly assessed. In this way he produced numerous new cultivated varieties of plums, lilies, and many other ornamentals and fruits.

The works of Charles Darwin, particularly The Variation of Animals and Plants under Domestication, greatly influenced Burbank. However his success in varying plant characters reinforced his belief in the inheritance of acquired characteristics, even though he knew of Gregor Mendel's research.