The influences of hydrolysis time, pH, temperature, enzyme and time on enzymatic hydrolysis of Polyrhachis vicina Roger protein by alcalase 2.4L were investigated. The antioxidant activity of the product was evaluated by determining five indexes including the scavenging activities of superoxide radical, DPPH? and hydroxyl radical, reducing power and inhibitory ability against oxidation of linoleic acid. By response surface methodology (RSM), the hydrolysis conditions were optimized to achieve the highest antioxidant activity of the product. Verification experiments confirmed the accuracy of equations more than 95%. Ultrafiltration and chromatography techniques were performed to purify enzymatic hydrolysates. It was found that peptides in the range of 5~10 kDa and 1~3 kDa mainly accounted for the activity. Basic peptides had greater scavenging capacity against superoxide radical and hydroxyl radical, as well as inhibitory ability against oxidation of linoleic acid, than acidic peptides. Acidic peptides had higher DPPH? radical scavenging activities and reducing power compared with basic peptides.