Abstract: Objectives: This study was to optimize the extraction and purification process parameters of donkey serum albumin (DSA) from Asini Corii Colla, to compare the functional properties of DSA before and after purification, and to identify the purity and antioxidant activity of purified DSA. Methods: In this study, Asini Corii Colla was used as raw material and DSA extraction rate was used as the response value, the optimum extraction process of salting-out-ultrasonic assisted extraction was determined by single factor and Box-Behnken response surface design. The concentration of DSA in Asini Corii Colla was purified by Sephadex G-75, and the purity of DSA in purified Asini Corii Colla was identified by SDS-PAGE gel electrophoresis. The water holding capacity, oil holding capacity, emulsification and foaming properties of DSA in Asini Corii Colla before and after purification were compared. Finally, DSA was used to analyze its antioxidant activity in vitro by scavenging ability of superoxide anion radical, ABTS⁺ radical, DPPH radical and hydroxyl radical. Results: The optimum extraction process parameters of DSA in Asini Corii Colla were as follows: the ratio of solution was 1:11 g/mL, the ratio of material to liquid was 1:8.3 g/mL, the ultrasonic power was 400 W, and the ultrasonic time was 31 minutes. Under these conditions, the extraction rate of DSA was 49.57%, and the purity of DSA after purification could reach 83.7%. The results of functional properties showed that the water holding capacity, oil holding capacity, emulsification and foaming properties of DSA purified under different pH conditions were enhanced compared with those before purification. The results of antioxidant activity in vitro showed that DSA had strong antioxidant activity in vitro. When the concentration of DSA was 10.0 mg/mL, the scavenging rates of superoxide anion radical, ABTS⁺ radical, DPPH radical and hydroxyl radical were 73.39%, 83.61%, 71.24% and 83.79%, respectively. Conclusions: The extraction, purification and identification techniques of DSA in Asini Corii Colla are simple, rapid, and high feasibility, which provides a certain reference for the high-value utilization of Asini Corii Colla and the development of functional food.