Abstract: In this study, the by-products of Chinese softshell turtle processing were used as raw materials, which provided the possibility to improve the extraction efficiency of Chinese softshell turtle oil and make it high-value utilization to provide a new source of profit for production enterprises. The extraction experiment of turtle oil was carried out by using ultrasonic combined with complex enzymatic hydrolysis (The pH of enzymatic hydrolysis was controlled at 7.5~8.5), and the extraction parameters were optimized by single factor experiments combined with orthogonal experiment, and the optimal extraction process conditions of ultrasonic-complex enzymatic hydrolysis method were obtained: The ratio of solid to liquid was 1:1.5 (g/mL), the amount of protease was 2.4%, the ratio of enzyme was 1:1.5 (neutral protease:alkaline protease), the enzymatic hydrolysis time was 2 h, the enzymatic hydrolysis temperature was 65 ℃, the ultrasonic power was 180 W, and the ultrasonic time was 30 min. The extraction rate of Chinese softshell turtle oil was 81.65%±0.62%. After the Chinese softshell turtle oil was refined, its physical and chemical indicators were all in line with the national aquatic industry standard (SC/T 3502-2016) refined fish oil first-class standard. At the same time, fatty acid composition analysis showed that the refined Chinese softshell turtle oil contained a total of 28 kinds of fatty acids, of which saturated fatty acids accounted for 23.81% of the total, monounsaturated fatty acids accounted for 46.94% of the total, polyunsaturated fatty acids accounted for 26.38% of the total, DHA and EPA content accounted for 10.05% of the total.