Abstract: The flavor precursor-enzymalysis liquor was prepared by the stepwise biological enzymolysis technology using Penaeus chinensis waste as raw material. Based on the preliminary hydrolysis of Penaeus chinensis waste by alcalase,enzymatic hydrolysis process of Penaeus chinensis waste with flavourzyme for the second time,was optimized by response surface methodology(RSM). In addition,the effects of single factors such as temperature,initial pH,and amount of flavoring protease on the degree of hydrolysis were investigated. Results showed that optimized enzymatic hydrolysis condition was:Ratio of material to water 1:9,flavourzyme amount 2.2%,primary pH6.4,temperature 59℃,and hydrolysis time 3 h. Under these conditions,the hydrolysis degree(DH)was 54.79%. The content of free amino acids in the hydrolysate was 33.6862 mg/mL,and the content of essential amino acids(such as threonine,lysine,isoleucine,phenylalanine,etc.)in the hydrolysiate was 27.2543 mg/mL. The contents of glutamate and lysine were obviously increased,and flavor strength had been greatly enhanced. Thus it was suitable for flavor seasoning. The high-quality Penaeus chinensis flavor base could be prepared by the double-enzyme stepwise hydrolysis,which had great significance to improve the utilization and overall value of Penaeus chinensis.