Abstract: Wheat gluten was enzymatically hydrolyzed by several commercially available proteases (Alcalase, Pepsin, Pancreatin, Neutrase) for the preparation of small peptides (<1000u) .The hydrolytic efficiency, the relationship between the hydrolysates hydrolysis degree and the polypeptide content, the molecular weight distribution and the amino acid content of hydrolysates, of these proteases on wheat gluten was compared.Subsequently, the relationships between the molecular weight distribution of hydrolysates and the polypeptide hydrolysates were further determined by animal experiments.The results showed that the four proteases had different enzymolysis effects on wheat gluten.In terms of total peptides content, the pepsin treatment group was the highest, followed by alkaline group.When the hydrolysates hydrolysis degree remained at a level of 12% of all four different proteases hydrolysates, the yield of small peptides (<1000u) was more than 80% for alkaline treatment group, around 65% for Pancreatin treatment group.Enzymatic hydrolyzates for both of the two groups could significantly promote digestive capability and immune function of experimental animals.Simultaneously, the total amino acid content of the four different enzymatic hydrolyzates was increased compared with the control group (without enzymolysis) , while the content of free amino acid had little difference, which demonstrated that wheat gluten was hydrolyzed moderately and no excessive enzymolysis existed.