The combination of different pretreatment methods can effectively overcome recalcitrance of lignocellulosic biomass to ensure its highly efficient conversion into bio-based products. In this study, the combined pretreatments of chemical methods (hydrothermal treatment and Fenton treatment) with mechanical refining were used to improve the enzymatic hydrolysis efficiency of poplar branches. The results indicated that hydrothermal pretreatment and Fenton pretreatment can effectively improve the enzymatic hydrolysis of poplar substrates, e.g., the maximum glucose conversion yield and glucose concentration reached 92.4% and 20.8 g/L, respectively. The pre-hydrolysates contained some valuable components such as monosaccharides, oligosaccharides, acetic acid, furfural, and hydroxymethylfurfural. The main characteristics (specific surface area, water retention value, fines content, and surface lignin concentration) of poplar substrates were obviously changed by the combined pretreatment, which benefit the enzymatic hydrolysis.