Abstract: To explore an efficient and economic method for enzymatic hydrolysis of silkworm pupa protein, using fresh pupa and dried pupa as raw materials, the effect of neutral protease and flavourzyme on the degree of hydrolysis and protein yield was examined under neutral condition. The results showed that fresh pupa could obtain higher protein yield and higher degree of hydrolysis than dried pupa. The protein yield of enzymatic hydrolysis using flavourzyme was higher than that of neutral protease, but its hydrolysis degree was lower. This might be caused by the low endonuclease activity of flavourzyme. So the fresh pupas were preferred in practice and proper protease should be used according to different requirements of protein hydrolysate.