Abstract: The onion allinase has been immobilized on chitosan micropheres with glutaraldehyd as cross-inking agent by absorption and cross-linking method. Some factors which had effects on the activity of immobilized onion allinase had been studied. The optimal conditions of immobilization was achieved by using the method of single factor experiment, amount of enzyme was 217.25U/g, adsorption time was 6h, glutaraldehyde concentration was 0.50%, cross-linking time was 3h, reaction temperature was 35℃ , reaction time was 10min. The recovery rate of immobilized onion allinase activity was above 50% , when immobilized enzyme was repeatedly used for 7 times.