Abstract: In order to reduce the risk of silkworm allergic reaction, to improve the use of high-quality protein and the preparation of immunoactive peptide, the enzymatic hydrolysis of silkworm pupa protein was optimized. For the preparation of immunoactive peptide, the five kinds of enzymes ( bromelain, alkaline protease, neutral protease, flavourzyme, papain) were selected to hydrolyze the defatted silkworm pupa protein. By the detection of spleen cell proliferation and the degree of hydrolysis, the optimal enzyme were determined.On the basis of that, the optimal condition was identified with four singe-factors of hydrolysis ( E0/S0, p H, temperature and substrate concentration) .The results showed that alkaline protease was the optimal enzyme and the optimum conditions of defatted silkworm pupa protein by alkaline protease enzyme was as that of the amount of enzyme = 6.0%, temperature = 55 ℃, enzymolysis time = 2.00 h, p H = 8.0, water∶ substrate = 20∶ 1.The degree of hydrolysis was19.96% ± 1.02% and immunoreactive OD490 nmwas 0.2512 ± 0.0125.