Abstract: This paper deals with the optimization of the process conditions for the enzymatic hydrolysis of horse bone marrow protein using response surface methodology. Taking the concentration of horse bone marrow protein and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability as indicator, single factor test were conducted with extraction temperature, extraction time, solid-liquid ratio, and extraction times as experimental factors, then the response surface method was used to optimize the extraction process and select the optimal extraction conditions. Meanwhile, the antioxidant activity of horse bone marrow protein was evaluated under the determination of DPPH, hydroxyl (·OH), superoxide free radical (\rmO_2^- \cdot ) and 2, 2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate (ABTS) free radicals scavenging ability and total reduction ability. The results showed that the optimal extraction condition for horse bone marrow protein was: extraction temperature 45℃, extraction time 2 h, solid-liquid ratio 1:20 g/mL, extraction times of 2. The concentration of horse bone marrow protein was up to 70.40 mg/mL, DPPH free radical scavenging activity was 68.92%, and its comprehensive score was 91.017%. The horse bone marrow protein exhibited good antioxidant capacity, and its half-inhibitory concentration (IC₅₀) for scavenging DPPH, ·OH, \rmO_2^- \cdot and ABTS radicals are 0.061, 0.067, 0.250 and 0.290 mg/mL, respectively. This study could provide a theoretical basis for the further development and application of horse bone marrow protein.