Lactoglobulin, one of major allergens in cow milk, was studied in this paper, and the T cell immune tolerance hydrolysates of β-lactoglobulin were prepared and identified to provide a theoretical basis for oral immunotherapy for patients with cow milk allergy. Firstly, the bioinformatics method was used to predict the possible β-lactoglobulin T-cell epitopes, and then, β-lactoglobulin was respectively hydrolyzed by six kinds of protease. The amino acid sequences of polypeptide of β-lactoglobulin hydrolysates were analyzed by mass spectrometry,and these peptides were synthesized by solid phase synthesis. The immune tolerance of the peptides were identified by T cell proliferation test. Therefore, trypsin, protamex, papain and neutrase hydrolysates were initially screened for T cell immune tolerance. Furthermore, the immune tolerance of β-lactoglobulin hydrolysates were evaluated by animal experiments. The levels of specific antibody (IgE, IgG₁, IgG_2a), Th1 cytokine (IFN-γ, IL-17), Th2 cytokine (IL-4, IL-5, IL-13), histamine, chitinase-3-like protein 1 in mice serum and the differentiation of mice spleen cell subsets were measured in vivo. The results showed that trypsin, protamex and papain hydrolysates had oral tolerance, among which protamex and papain hydrolysates had been reported innovatively. Although neutrase protease hydrolysates contained T cell epitopes, they were still allergenic. Therefore, the hydrolysates containing T cell epitopes did not necessarily have oral immune tolerance and need to be verified in vivo. The conclusion of this study could provide an important theoretical basis for the development of new anti-allergy milk-based materials and clinical treatment of milk allergy.