Abstract: Objective: To screen the optimal extraction temperature and optimal enzymatic hydrolysis process of deer bone protein and detect its antioxidant activity. Method: According to the protein concentration of deer bone protein, the optimal extraction temperature was selected. According to the degree of hydrolysis (DH), the optimal enzymatic hydrolysis process of deer bone polypeptide was screened through single factor and response surface experiment design, and the antioxidant activity of deer bone polypeptide was evaluated by measuring DPPH· and ·OH scavenging ability of deer bone polypeptide. Results: The optimal extraction temperature was 95 ℃. The optimal digestion process for pepsin and trypsin was determined by response surface method optimization and actual verification as the amount of pepsin enzyme 6200 U/g, temperature 37.3 ℃, pH2.0, and time 3.2 h, the degree of hydrolysis at this time was 11.23%; the amount of trypsin was 6300 U/g, the temperature was 37.2 ℃, the pH was 8.1, and the time was 4.0 h, the degree of hydrolysis at this time was 23.09%. Deer bone polypeptide had scavenging ability to DPPH· and ·OH free radicals, and its IC₅₀ values were 3.72 and 2.24 mg/mL, respectively. Conclusion: The best extraction temperature and optimal enzymatic hydrolysis process of deer bone protein are obtained. Deer bone protein has a good scavenging ability on DPPH· and ·OH free radicals, indicating that deer bone polypeptide has good antioxidant activity.