The ameliorative effect of soft-shell turtle peptides (STP) on oxidative damage in mice was evaluated. DPPH radical scavenging rate was used as the evaluation indicator to optimize the separation and purification process using the ion-exchange resin method to obtain high-activity antioxidant STP. A D-galactose (D-gal)-induced oxidative damage model was established in mice, and assay kits were used to measure superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content. The results showed that the gel cation-exchange resin (GC) was the best for the separation and purification of antioxidant peptides. When the static adsorption pH was 1.0 and ethanol volume fraction of the desorption solution was 50%, the DPPH radical scavenging rate of GC increased by 34.42% (P<0.001) compared with that of unpurified STP. Compared to the model group, in the GC-purified STP (GC-STP) group, the SOD activity in the liver and thymus tissues significantly increased by 62.28% (P<0.05) and 59.18% (P<0.01), respectively; the GSH-Px activity in the liver, spleen and thymus tissues significantly increased by 32.52% (P<0.05), 7.50% (P<0.05), and 178.96% (P<0.01), respectively; and the MDA content in the liver tissue significantly decreased by 28.28% (P<0.05). Therefore, GC-STP could effectively ameliorate D-gal-induced oxidative damage of liver tissue in mice. This study can serve as a theoretical foundation for improving oxidative damage using GC-STP.