Abstract: The bioactive peptide was prepared by enzymatic hydrolysis of pearl gentian grouper meat. Based on the degree of hydrolysis and DPPH free radical scavenging rate,the response surface methodology was used to optimize the preparation process based on the single factor experiment. And the ultra-filtration method was used to isolate and purify the enzymatic hydrolysis products,also to investigate the antioxidant activity. The results showed that the enzymatic hydrolysis conditions of the pearl gentian grouper were as follows:The flavor protease was used,the enzyme added amount was 1050 U/g,and the enzyme was hydrolyzed for 5.5 h under the conditions of pH7.0,53℃ and the ratio of material to water 1:3.5,the degree of hydrolysis was 9.99%±0.39%. Both the enzymatic hydrolysis products and ultrafiltration components had strong DPPH free radical scavenging ability,and their IC₅₀ values were between 0.63~0.88 mg/mL;EH-2(5~8 kDa)and EH-3(3~5 kDa)had strong scavenging ability of hydroxyl radicals,and its IC₅₀ values were 16.94 mg/mL and 16.38 mg/mL,respectively. The enzymolysis products and ultrafiltration components had reducing ability,and the enzymolysis products had the best reducing ability. The optimized enzymatic preparation process of peptides from pearl gentian grouper was reasonable and feasible,and its enzymatic hydrolysis products and ultrafiltration components had strong antioxidant properties and can be used as the base material of functional food.