Pregl, who was born at Laibach (now Ljubljana in Slovenia), was the son of a bank official. He graduated in medicine from Graz (1893) where he became an assistant in physiological chemistry in 1899. In 1910 he became head of the chemistry department at Innsbruck, remaining there until 1913 when he returned to Graz to become director of the Medico-Chemical Institute.

Pregl began research on bile acids in about 1904 but soon found that he could only obtain tiny amounts. This led him to pioneer techniques of microanalysis. Justus von Liebig had needed about 1 gram of a substance before he could make an accurate analysis; through his new techniques Pregl was capable of working with 2.5 milligrams. This was achieved by the careful scaling down of his analytic equipment and the design of a new balance, which was produced in collaboration with the instrument maker W. Kuhlmann of Hamburg. With this he was capable of weighing 20 grams to an accuracy of 0.001 milligram.

The techniques developed by Pregl are of immense importance in organic chemistry and he was awarded the Nobel Prize for chemistry in 1923 for this work.