This article aims to analyze the effects of enzyme treatment concentration, temperature, and time on peanut protein so as to obtain an optimal enzymatic hydrolysis condition for flavorzyme (Fla) and alkaline protease (Alk). The results were as follows: enzymatic hydrolysis temperature 60 °C and 55 °C, enzyme concentration 10% and 4%, enzymatic hydrolysis time 80 min and 60 min, and double enzyme hydrolysis ratio 2% Fla + 5% Alk, respectively. The BALB/c mice were sensitized with gavage of peanut protein before and after enzyme treatment to evaluate the effects of different enzyme treatments on peanut allergenicity. Compared with the mice sensitized with raw peanuts, the weight growth rate of the mice sensitized with enzyme treatment peanut increased but not as much as the control, the degranulation degree of mast cell and basophils decreased, the inflammatory infiltration and congestion in jejunum and lung tissue decreased, the expression of proinflammatory factors and thymic stromal lymphopoietin (TSLP) gene decreased, and the secretion of specific antibodies (IgE, and IgG) decreased, and the binding ability of peanut protein with peanut-specific IgE antibodies decreased as well. The results above indicate that the allergenicity of peanut protein decreases after enzyme treatment and the dual enzyme (Fla + Alk) treatment can be much more efficient.