To optimize the enzymatic conversion of widely available lignocellulosic biomass-wheat straw (WS), pretreatment facilitating the enzymatic saccharification process was first performed by alkaline peroxide, resulting in a substrate consisting of 60.17% cellulose, 29.53% hemicelluloses, and 4.59% lignin. Using response surface methodology, the combined effects of enzyme loading, substrate concentration, surfactant concentration, and reaction time on hydrolysis yield from enzymatic saccharification of WS were further investigated. The results showed that both enzyme loading and substrate concentration had interactions with surfactant concentration. A quadratic polynomial equation for predicting the hydrolysis yield was developed. The experimental results were in good agreement with predicted values. Therefore, the model could be successfully used to identify the effective combinations of the four factors for predicting hydrolysis yield.