Abstract: The effects of enzymatic hydrolysis time, temperature, and amount of enzyme on the removal of hydroxyl radical from squid muscle were investigated.The enzymatic technology of squid protein was optimized by Box-Behnken design and response surface methodology ( RSM) .In the meantime, the antioxidant activities of squid antioxidant peptides were studied by using free radical scavenging and antioxidant activity in mice. The results showed that the hydroxyl radical scavenging rate was significantly affected by time and amount of enzyme. The optimum conditions were enzymatic hydrolysis time 6 h, amount of enzyme 0.07 g/10 g, enzymolysis temperature 55 ℃, under which the hydroxyl radical scavenging rate was 90.31% ± 0.54%.The EC50 of antioxidant peptide from squid against DPPH radical, hydroxyl radical and superoxide anion radical were 8.8, 16.5mg/m L and 10.3mg/m L, respectively.SOD activity of Squid antioxidant peptide in the high and low dose group mice was higher than the control group of 19.2% and 14.2%.GSH activity of Squid antioxidant peptide in the high and low dose group mice was higher than the control group of 23.4% and 14%. Squid antioxidant peptides had strong antioxidant activity through antioxidant experiments in vitro and in vivo.