In this study, the effect of dual-enzyme hydrolysis on the allergenicity of the major proteins in skimmed milk was evaluated based on the degree of hydrolysis, molecular mass distribution, immunoglobulin G (IgG) binding capacity and allergic patients’ serum IgE binding capacity of hydrolysates. The results showed that after one-step hydrolysis for 80 min at an enzyme/substrate ratio of 3000 U/g and an Alcalase to Flavourzyme ratio of 3:1, the IgG binding capacity of casein, α-lactalbumin and β-lactoglobulinin in skimmed milk decreased significantly by 74.93%, 97.24% and 93.46%, respectively (P < 0.05).The advanced structure of proteins was damaged, leading to an increase in the α-helical and β-turn contents, and a decrease in the β-sheet content. In addition, the free sulfhydryl content increased significantly to 14.29 μmol/g (P < 0.05), the surface hydrophobicity also increased, and the structure became loose. The particles in skimmed milk were aggregated, causing the average particle size to increase significantly to 346.90 nm (P < 0.05) and reducing the stability of skimmed milk. The binding capacity to serum IgE from patients with milk allergy of the enzymatic hydrolysate decreased significantly by 23.53% (P < 0.05), and the overall allergenicity decreased significantly by 69.60% (P < 0.05). Therefore, dual-enzyme hydrolysis is an effective method to reduce the allergenicity of skimmed milk.