The present study demonstrated the hydrolysis of peony seed protein isolate (PSPI) by using alcalase and resulted in the generation of an anti-oxidative peptide. In brief, a model was used to illustrate the enzymolysis of PSPI with the determination of kinetic factors as per investigation information. The model proved suitable to explain the PSPI hydrolysis by alcalase. A novel anti-oxidative peptide was obtained successfully by ultrafiltration and a series of chromatography techniques. Subsequently, a purified fragment was identified with the amino acid sequence of SMRKPPG followed by its synthesis and evaluation of its anti-oxidative activities. After hydrolysis, the peony seed protein hydrolysate (PSPH) with the degree of hydrolysis of 18% displayed the most significant antioxidant action which was further used to isolate the anti-oxidative peptide.