Born in Leiden, Netherlands, Kolff was the eldest of a family of 5 boys. Kolff studied medicine in his hometown at Leiden University, and continued as a resident in internal medicine at Groningen University. One of his first patients was a 22-year-old man who was slowly dying of chronic kidney failure. This prompted Kolff to perform research on artificial renal function replacement. Also during his residency, Kolff organized the first blood bank in Europe (in 1940). Kolff's first prototype dialyzer was developed in 1943, built from orange juice cans, used auto parts, and sausage casings. Over a two-year span, Kolff had attempted to treat 15 people with his machine, but all had died. In 1945, Kolff successfully treated his first patient, a 67-year-old woman, from kidney failure using his hemodialysis machine.
During World War II, he was in Kampen, where he was active in the resistance against the German occupation. Simultaneously, Kolff developed the first functioning artificial kidney. He treated his first patient in 1943, and in 1945 he was able to save a patient's life with hemodialysis treatment. In 1946 he obtained a PhD degree summa cum laude at University of Groningen on the subject. It marks the start of a treatment that has saved the lives of millions of acute kidney injury or chronic kidney failure patients ever since.