Whey protein, a by-product of cheese processing, is ubiquitously applied in infant formula. Nevertheless, it contains β-lactoglobulin, an allergenic component that can be enzymatically hydrolyzed to destroy its allergenic epitopes and antigenicity. The whey protein hydrolysate (WPH) obtained through enzymatic hydrolysis exhibits a wide range of biological activities. However, its utilization in preventing whey protein food allergies has received limited research attention. This study aimed to examine the preventive effect of WPH intervention on whey protein-induced food allergy in BALB/c mice. The results showed that WPH intervention notably mitigated the development of allergic reactions in mice with whey protein-induced food allergies. The intervention with WPH significantly reduced the symptom score of allergic reactions in mice with whey protein-induced food allergies (39.38%, P < 0.01). Early intervention with WPH also led to a significant reduction in the serum levels of antibodies and related cytokines, including IgE, histamine, IgG, and monocyte chemotactic protein-1 (MCP-1) in mice (P < 0.05). A potential mechanism for alleviating allergic reactions was identified from the proteomic findings. WPH was found to upregulate the Th1 differentiation pathway and IgA secretion pathway by increasing MHC II protein expression, thereby alleviating allergic reactions to whey protein in food. Regarding the gut microbiome, WPH intervention led to a decrease in the relative abundance of harmful bacteria, including Prevotellaceae_UCG-001, and Alloprevotella (5.4% and 2.3%, respectively; P< 0.05). It increased the relative abundance of beneficial bacteria such as Turicibacter, Roseburia, and Alistips (3.8%, 0.6%, and 1.4%, respectively; P < 0.05). The present study suggests early WPH intervention may attenuate whey protein-induced food allergic reactions.