The research aimed to establish a kind of effective and practical method to produce aprotinin. The trypsin was covalently linked with chemical modified chitosan and used to purify aprotinin from the extract of cattle lungs by affinity chromatography. The result shows that the specific activity of immobilized trypsin on chitosan is 71428BAEE/mg, and the activity recovery of trypsin is 73.5%. It is accepted as a stable and low-cost method and suitable for purifying aprotinin with high activity in industrial manufacturing.