The large yellow croaker liver was used as the raw material, and the optimal protease was used to extract and extract the large yellow croaker liver oil. Taking the large yellow croaker liver oil extraction rate as an evaluation index, the best enzymatic extraction process was determined through single-factor experiments and response surface experiments. The extraction rate, quality, and fatty acid composition of the large yellow croaker liver oil were compared with those obtained by light alkali method. The results showed that the best enzymatic extraction conditions were as followed: for large yellow croaker liver was using neutral protease, the ratio of material liquid ratio was 1∶2, the amount of enzyme added was 2.5%, the value of pH was 7.3, the hydrolysis time was 4 h, and the hydrolysis temperature was 50.3 ℃. Under the conditions, the extraction rate of large yellow croaker liver oil was 78.39%, and the quality was better than that of light alkali method. The oil was clear, the acid value was (5.83±0.15) mg/g, the iodine value was (142.65±0.22) mg / 100 g. There were 13 kinds of fatty acids, 5 more than that of light alkali method. There are 8 kinds of unsaturated fatty acids, including 19.71 g/100 g saturated fatty acids, 62.63 g/100 g monounsaturated fatty acids and 17.62 g/100 g polyunsaturated fatty acids. The extraction rate, quality and fatty acid composition of liver oil from large yellow croaker by enzymatic method were better than those by alkali method.