Abstract: In order to improve the comprehensive utilization rate of walnut, the technology of synchronous producing oil and polypeptide from walnut by aqueous enzymatic method was optimized, and the fatty acid composition of the oil was analyzed. By comparing the effects of four different proteases and cellulase on oil extraction rate and polypeptide yield of walnut, the optimal enzyme combination was then determined. On this basis, the effects of pH, enzymolysis temperature, enzymolysis time, solid-liquid ratio and dosage of enzyme on the oil extraction rate and the yield of polypeptide from walnut were studied by single factor and L₁₈(3⁵) orthogonal experiment, and the optimal extraction conditions were finally obtained. The fatty acid composition of walnut oil was analyzed by gas chromatography. The results showed that the combination of papain and cellulase (2:1, w/w) was the best enzyme combination. The optimal synchronous extraction process of oil and polypeptide were as follows: Dosage of enzyme 3.0%, solid-liquid ratio 1:5 (g/mL), pH5, enzymolysis time 3.0 h, enzymolysis temperature 60 ℃. Under these conditions, the oil extraction rate of walnut could reach 53.37%, and the yield of polypeptide was 4.01 mg/g. The determination results of gas chromatography indicated that five fatty acids were detected in walnut oil, including linoleic acid (62.26%), oleic acid (18.64%), α-linolenic acid (10.57%), palmitic acid (6.00%), stearic acid (2.53%). Walnut oil was rich in unsaturated fatty acids. The total content of unsaturated fatty acids in walnut oil was up to 91.47%, in which the contents of polyunsaturated fatty acids and monounsaturated fat acids were 72.83% and 18.64%, respectively. The process could provide reference for the industrial application of synchronous producing oil and polypeptide from walnut by aqueous enzymatic method.