Abstract: Objective: To investigate the technological conditions and antioxidant properties of Asini Corii Colla (ACC) peptides prepared by enzymolysis. Methods: The enzymatic hydrolysis conditions of ACC protein peptide prepared by Alcalase 2.4L were optimized using ACC as raw material. On the basis of single factor experiments, orthogonal test was carried out to determine the optimum process conditions with pH, enzyme-substrate ratio and enzymolysis temperature as the factors and the degree of hydrolysis (DH) of ACC as the indexes. To comprehensively analyze the enzymatic hydrolysis effect, the molecular weight distribution of ACC solution before and after enzymatic hydrolysis was determined by high performance gel chromatography, and the antioxidant activity of ACC solution before and after enzymatic hydrolysis was investigated by detecting scavenging rates of ABTS⁺ and DPPH free radicals. Four kinds of ultrafiltration membranes with different pore sizes were used to separate the ACC peptide solution, and the antioxidant activity of ACC peptide with different molecular weight was studied. Results: The optimum enzymolysis conditions were pH10.5, enzyme-substrate ratio 9% and enzymolysis temperature 63 ℃. Under these conditions, the DH was 15.93%. After enzymatic hydrolysis, the molecular weight of ACC solution was mostly below 5000 Da. When the concentration was 10 mg/mL, the ABTS⁺ free radical scavenging rate of the ACC peptide solutions before and after enzymolysis were 75.83%±0.32% and 93.16%±0.21%, respectively. The ability of scavenging DPPH free radicals was 16.93%±2.41% and 39.95%±1.27%. Five groups of ACC peptides with different molecular weight (>30 kDa, 10~30 kDa, 3~10 kDa, 1~3 kDa and <1 kDa) were obtained by ultrafiltration. It was showed that the ACC peptides with small molecular weight had good antioxidant effects. Conclusion: Alcalase 2.4L could be effectively used for preparing CCA peptides, and the ACC peptides possess good antioxidant activities. These results would provide scientific basis for further research on the antioxidant mechanism of ACC peptide.