In this study, casein was hydrolyzed with seven food-grade proteases, including flavourzyme, trypsin, chymotrypsin, alkaline protease, papain, bromelain, and neutral protease and the allergenicity of the resultant hydrolysates was assessed using a serum pool of patients with milk allergy. Moreover, the molecular mass distribution, degree of hydrolysis and residual epitope information were evaluated in order to select the most suitable protease for the production of casein products with reduced allergenicity. The results showed that under the same conditions, the binding ability of casein to specific antibodies decreased to the greatest extent after being treated with chymotrypsin or flavourzyme, so they could be used as a candidate protease for the preparation of subsequent hypoallergenic casein hydrolysates. The results of T-cell epitope prediction and analysis of casein hydrolysates showed that only one T-cell epitope peptide consisting of more than nine amino acids LHSMKEGIHAQQK remained after chymotrypsin hydrolysis of casein.