In order to prepare the hypophthalmichthys molitrix skin peptides with good antioxidant activity, the hypophthalmichthys molitrix skin was hydrolyzed by using of the four common commercial enzymes, namely, trypsin, Alcalase 2.4L, bromelain and papain; ABTS radical scavenging activity and ferrous ion-chelating activity were determined; and the hydrolyzates were separated by ultrafiltration and gel chromatography to obtain better hydrolyzate separation fractions. The results showed that the antioxidant activity of the protein hydrolysate increased, among which the antioxidant activity of the protein hydrolysate after a 2-hour enzymolysis had a stronger antioxidant activity. An ultrafiltration was carried out to the hollow fiber ultrafiltration membranes with the cut-off molecular weight of 10 kDa, 5 kDa and 3 kDa. Among the 4 fractions after the ultrafiltration., the smaller the molecular weight was, the stronger the antioxidant activity was. The fraction with a molecular weight less than 3 kDa was separated into 3 sub-fractions by Sephadex G-15 gel chromatography, among which the larger the molecular weight was, the stronger the antioxidant activity was, whose ABTS radical scavenging ability (79.65±0.87)% and Fe2+ chelating capacity (93.40±0.20)% were determined at the concentration of 0.51 mg/mL. This study provided a theoretical reference for the exploitation and utilization of antioxidant peptides from hypophthalmichthys molitrix skin.