Objective: This study aimed to optimize the extraction process of visceral fish oil from grass carp. Methods: Using grass carp viscera as raw material, fish oil was extracted by using ultrasonic assisted enzyme method. The effects of enzyme type, enzyme addition amount, enzymatic hydrolysis time, enzymatic hydrolysis temperature, ultrasonic treatment time, and ultrasonic treatment power on the extraction rate of fish oil were investigated. Results: The optimal process conditions for ultrasonic assisted enzymatic extraction of fish oil were neutral protease hydrolysis, enzyme addition of 2.5%, enzymatic hydrolysis time of 2 hours, enzymatic hydrolysis temperature of 45 ℃, ultrasonic treatment power of 50 W, and ultrasonic treatment time of 30 minutes. At this time, the extraction rate of fish oil was 68.4%. A total of 27 fatty acids were detected by GC-MS, including 10 saturated fatty acids, accounting for 21.12% of the total fatty acid content, mainly palmitic acid and stearic acid; There were 7 kinds of monounsaturated fatty acids, accounting for 54.68% of the total fatty acids, mainly oleic acid, palmitoleic acid and eicosanoic acid; There were 10 kinds of polyunsaturated fatty acids, accounting for 24.20% of the total fatty acids, mainly linoleic acid. Conclusion: Compared with individual enzymatic extraction, ultrasonic assisted enzymatic extraction of grass carp visceral fish oil significantly improved the yield of fish oil extraction.