The technology of enzymatic extraction of fish oil has many advantages, such as moderate operating conditions, lower energy consumption and high efficiency. Moreover, it could reduce the breakage for the functional component of fish oil. In enzymatic extraction of oil, the investigation of the property of enzymatic reaction is propitious to improve the enzymolysis efficiency. In this study, the 1398 neutrase was used for hydrolyzing fish protein, then analyzed the impacts to the enzymolysis efficiency which were induced by the different initial substrate concentration and different initial enzyme concentration, the result showed that the higher substrate concentration generate inhibition to the enzyme. And then, the properties of enzymatic hydrolysis were studied by the Michaelis-Menten equation of substrate inhibition and the enzymatic hydrolysis kinetics equation which is derived by theory. By means of the verification to the enzymatic hydrolysis kinetics model, it could see that the model in line with the actual situation better at a lower degree of hydrolysis. Lastly, the critical enzyme concentration and critical substrate concentration of enzymatic reaction could be obtained by deducing the enzymatic hydrolysis kinetics model.