The trypsin was covalently linked with chemical modified granulechitosan and was used to isolate and purify aprotininum from the extract of cattle lungs by affinity chromatography. Then high-purity aprotininum was prepared after ultrafiltrating and freeze drying. The result showed:the specific activity of immobilized trypsin on chitosan was 25 950 kU/g，60.5％protein was coupled，and the activity reccvery of trypsin was 55％. The purity of aprotininum was high，and the activity reccvery of trypsin on immobilized trypsin had low non specific adsoption and ideal anti-contemination，and it could be used more than 72 times. It was accepted as a simple and stable method and suitable for purifying aprotininum with high activity in industrial manufacturing.