Abstract: To select the superior protease for enzymatic hydrolysis of squid viscera, hydrolyzate of squid viscera and its DPPH free radical scavenging capacity were analyzed, and the distribution of molecular weight was determined. Alkaline protease, neutral proteinase, trypsin, animal protease, flavored proteinase, and papain were selected for enzymatic hydrolysis of squid viscera. Hydrolysis degree, nitrogen yield and hydrolysis yields of different hydrolysate were compared by measuring total nitrogen content and amino nitrogen content of the hydrolysate. Tryptic hydrolyzate was eluted with water, 20%, 60% and 80% methanol-water through macroporous resin HP-20, and then analyzed by LC-MS. The results showed that the molecular weight of short peptide in the hydrolysate was 100-2400. The samples processed by 60% methanol-water gradient was more concentrated and the molecular weight of the hydrolysates were mainly about 200. The analysis of DPPH free radical scavenging capacity of each elution gradient hydrolyzate indicated that DPPH radical scavenging capacity of the hydrolyzate eluted with 60% methanol-water was 40.21%. The superior protease for enzymatic hydrolysis of squid viscera was trypsin, and 60% methanol-water was the optimum elution concentration.