Abstract: The enzymatic hydrolysis parameters for preparation of antioxidant peptide from seahorse protein were optimized. Base on the results of single factor experiment, content of free amino acids, reducing power and scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were chosen as the measure indexes, the influences of enzyme species, pH value, enzymolysis temperature, reaction time and enzyme in solid matter for enzymatic hydrolysis were inspected. Optimum hydrolysis conditions were confirmed as enzyme in solid matter, 6000 U/g; enzymolysis temperature, 45℃; and reaction time, 9 hours. Content of the free amino acids, reducing power and scavenging activity on DPPH radical under optimum conditions was 4.198 mg/mL, 92.758% and 1.091, respectively. The optimum hydrolysis conditions were generated and laid a solid foundation for the development of health care products.