The Fenton reaction has been widely used in the pretreatment of lignocellulose. It offers the advantages of simple operation, fast reaction speed, and low pollution. In this study, the effects of different proportions of Fenton reagents on the enzymatic hydrolysis of poplar were compared and analyzed, and the optimal ratio of Fenton reagents was obtained. The maximum yield of enzymatic hydrolysis of glucose in Fenton pretreatment samples was 406 mg/g at H2O2 and Fe2+ concentrations of 1.0 mol/L and 0.01 mol/L, respectively, which was 2.5 times that of untreated samples. Meanwhile, the composition analysis and FT-IR analysis showed that Fenton pretreatment could degrade lignin and hemicellulose effectively. X-ray diffraction (XRD) analysis showed that Fenton pretreatment can partially destroy the amorphous region of poplar. These findings will contribute to efforts to improve the viability of the Fenton pretreatment process for converting biomass into energy.