Abstract: The use of lipase in enzymatic transesterification to improve the content of omega-3 functional fatty acids in fish oil triglycerides has been a hot topic in food and feed industry in recent years. Accurate determination of the enzyme activity is of great significance to the application of lipase in fish oil industry. With 8 different commercial lipases as raw materials, the lipase activity detection method was studied from two aspects including the enzymatic hydrolysis activity and transesterification activity, and then applied to fish oil hydrolysis reaction and transesterification reaction. When the standard deviation of enzyme activity determination was used as an indicator, the results showed that polyvinyl alcohol-olive oil emulsification method was more applicable for hydrolytic activity than the p-nitrophenol method, while the octanoic acid-glycerol trioleate transesterification method was more applicable for transesterification activity than the p-nitrophenol method. The correlation coefficients of the lipase transesterification activity and the hydrolytic activity, fitted by linear model, power model and exponential model, were 0.07, 0.15 and 0.20, respectively, showing no significant correlation between these two activities and they cannot be predicted by each other. When the two lipase activity detection methods were used to screen high-enzymatic fish oil hydrolysis and transesterification lipases, the acid value of the fish oil hydrolysate of lipase L5 was 137.61 mg KOH/g, and the total contents of EPA and DHA from transesterification products of immobilized enzyme L8 was 55.38%. This study provides a more accurate determination of enzyme activity for the application of lipase in fish oil industry.