Abstract: In order to optimize the enzymolysis conditions of Katsuwonus pelamis viscera by protease and improve the utilization rate of Katsuwonus pelamis,the viscera of Katsuwonus pelamis was used as the raw material,and the fish oil extraction rate was used as the evaluation index. The Katsuwonus pelamis fish oil was extracted by bio-enzymatic technique through screening the best enzymes. The effects of pH value,enzymatic hydrolysis time,enzymatic hydrolysis temperature,liquid-solid ratio and enzyme addition amount on fish oil extraction rate in carp were determined through single factor and response surface experiment. The results showed that the extraction rates of fish oil from the viscera of carp were increased in different degrees by trypsin,alkaline protease,pepsin,papain and flavor protease. The extraction rate of visceral fish oil treated with alkaline protease was the best,reaching 57.46%. In addition,the fish oil in the Katsuwonus pelamis viscera had the highest fish oil extraction rate of 58.49%±0.45% under the conditions of pH8.40,enzymatic hydrolysis time 5.5 h,enzymatic hydrolysis temperature 55 ℃,liquid-solid ratio 1:1 and enzyme addition 2.0%. This study provided basic data and theoretical basis for the further development and comprehensive utilization of Katsuwonus pelamis waste.