The Lophius litulon liver was used as raw material for the extraction of fish oil via various extraction methods. The extraction rate by water extraction, potassium hydroxide (KOH) hydrolysis and protease hydrolysis were compared and the results revealed the protease hydrolysis extraction had a higher extraction rate with good protein-lipid separation as observed by optical microscope. Furthermore, subsequent experiments determined neutrase to be the best hydrolytic enzyme in terms of extraction rate and cost. The extraction conditions of neutrase hydrolysis were optimized by single-factor experiment and response surface analysis, and the optimal extraction rate was 58.40 ± 0.25% with the following conditions: enzyme concentration 2,000 IU/g, extraction time 1.0 h, liquid-solid ratio 1.95:1, extraction temperature 40.5℃ and pH 6.5. The fatty acids composition in fish oil from optimized extraction condition was composed of 19.75% saturated fatty acids and 80.25% unsaturated fatty acids. The content of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were 8.06% and 1.19%, respectively, with the ratio (6.77:1) surpassed to the recommendation in current researches (5:1). The results in this study suggest protease treatment is an efficient method for high-quality fish oil extraction from Lophius litulon liver with a satisfactory ratio of DHA and EPA.