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Bovine milk is one of the eight major allergenic foods identified by WHO/FAO, and β-lactoglobulin (BLG) is the primary allergen. Allergen standardization is very important for the diagnosis of milk allergy, and recombinant protein preparation is a promising method. In this study, BLG with high soluble expression was prepared by a method based on Escherichia coli. To verify the feasibility of recombinant BLG, the difference in physicochemical properties, allergenicity, and stability were investigated. Recombinant and natural BLG both had a size of approximately 230 nm with a zeta potential of -50 mV, indicating that they share the similar solubility and colloidal properties. Additionally, allergic mice model results confirmed that recombinant BLG showed comparable ability in stimulating antibody production, Th2 cytokine release, and cellular immunity compared with natural BLG. Then, the thermal stability of BLG was studied using spectrum analysis within the range of 25-85°C. Recombinant BLG showed satisfactory thermal stability, the concentration, protein structure and IgG binding capacity changed obviously until temperature reached at 55°C, although natural BLG is more heat resistant. After 28 days storage at 4°C in the form of lyophilized powder, there was no significant change in the content of recombinant and natural BLG. In summary, the physicochemical properties, allergenicity, and stability of recombinant BLG were comparable to those of natural BLG. This study provides theoretical reference for the development of standard bovine allergen, and it is expected that recombinant allergen can be used in the research and diagnosis of bovine milk allergy.