The compound peptide was used to digest the Euphausia superba to obtain the shrimp powder polypeptide, which was separated and purified by ultrafiltration separation and high-speed countercurrent chromatography to test its antioxidant activity in vitro. The results showed that the peptides isolated by ultrafiltration had the best antioxidant activity when the molecular weight ranged from 5 to 10 ku. When the concentration was 10 mg/mL, the superoxide anion radical scavenging rate reached 21.28%, and the DPPH free radical scavenging rate was 86.52%, the hydroxyl radical scavenging rate was 53.49%, and the Fe²⁺ chelation rate was 85.35%. High-speed countercurrent chromatography was carried out on peptides with a molecular weight ranging from 5 to 10 ku, using chloroform-butanol-methanol-water=4:0.75:3:2 as the two-phase solvent system, the lower phase as the stationary phase and the upper phase as the mobile phase; As for inversion, the speed was 900 r/min, the constant temperature water bath temperature was 25℃, the flow rate of the mobile phase was 1.5 mL/min, and the five components were separated under the injection concentration of 20 mg/mL. It was determined that when the concentration was 5 mg/mL, the ability of P3 component to scavenge superoxide anion radicals and Fe²⁺ chelation ability were significantly higher than the other four components, which were 37.18% and 92.33%, respectively. The free radical scavenging rate of P2 was the highest, which was 90.33%, and the P5 component had the highest hydroxyl radical scavenging rate of 70.11%. Antioxidant activity was significantly enhanced compared to the control group not isolated by HSCCC.