Abstract: Gelatin and chitosan-alginate gel were prepared by gelatin and modified gelatin respectively in this study, and the papain was encapsulated. The effects of gelatin-chitosan-sodium alginate concentration, CaCl₂ concentration and pH value on hardness and enzyme activity were studied by single factor test and uniform design. The optimum conditions for the preparation of immobilized enzyme were as follows:optimization results of using gelatin:0.45% gelatin, 0.42% chitosan, 2.55% sodium alginate, 0.8 mol/L CaCl₂, pH value was 7.5;Optimization results of using modified gelatin were as follows:0.45% modified gelatin, 0.15% chitosan, 2.55% sodium alginate, 0.8 mol/L CaCl₂, pH value was 6.5. The results of the uniform design showed that the enzyme activity using modified gelatin was 37% higher than that of gelatin, and the hardness was reduced by 35%. The activity of immobilized enzyme was improved by using modified gelatin, this study will lay a theoretical foundation for improving the activity of immobilized enzyme.