Abstract: In order to optimize the preparation process of donkey blood hemoglobin peptide and promote the in-depth development of donkey blood products, in this experiment, the process parameters of donkey blood total protein separation were determined by spectrophotometry. The scavenging ability of donkey hemoglobin on DPPH and hydroxyl（·OH） free radicals was determined to screen the best enzymatic hydrolysis enzyme. The enzymatic hydrolysis process of donkey hemoglobin was optimized by the response surface method. The results showed that the separation effect was best when centrifuged at 4 000 r/min for 15 min. Among the four proteases, alkaline protease had the strongest average scavenging ability of DPPH and hydroxyl radicals. The enzymatic hydrolysis process of donkey blood hemoglobin peptidation was 8 260 U/g, pH 9.79, enzymatic hydrolysis temperature of 55.75 ℃, and the scavenging rate of DPPH radicals reached 75.99%.