Ultrafiltration, macroporous resin, gel filtration chromatography and semi preparative liquid chromatography were used to separate and purify the enzymatic hydrolysate of Chinese roundabout meat. DPPH free radical scavenging rate, ABTS radical scavenging rate, iron reduction capacity (FRAP) and oxygen free radical absorbability (ORAC) were used as indicators to prepare antioxidant peptides with high purity and strong antioxidant activity. Enzyme activity and MDA production were measured. The results showed that the antioxidant activity of the enzyme hydrolysate after ultrafiltration was the strongest when the molecular weight was ＜1 kDa. Macroporous resin, gel filtration chromatography and semi preparative liquid chromatography were used to isolate the mixture containing antioxidant peptides (the purity of antioxidant peptide reached 90%). In vitro antioxidant activity showed that DPPH free radical scavenging rate, ABTS free radical scavenging rate and ORAC of the antioxidant peptide were the highest, and there was no significant difference between ORAC and glutathione (P＞0.05). Through the determination of antioxidant enzymes and MDA increase value in mice, except for GSH-Px, there was no significant difference between the test group and glutathione positive control group (P>0.05), indicating that the anti-inflammatory effect of this study was obtained. Oxidized peptide has the same antioxidative effect as glutathione, and has deep development and utilization value.