A major challenge in converting lignocellulose to biofuel is overcoming the resistance of the biomass structure. Herein, sequential dilute acid-alkali/aqueous ammonia treatment was evaluated to enhance enzymatic hydrolysis of poplar biomass by removing hemicellulose first and then removing lignin with acid and base, respectively. The results show that glucose release in sequential dilute acid-alkali treatments (61.4-71.4 mg/g) was 7.3-24.8% higher than sequential dilute acid-aqueous ammonia treatments (57.2-61.8 mg/g) and 283.8-346.3% higher than control (16.0 mg/g), respectively. Dilute acid treatment removed most hemicellulose (84.9%) of the biomass, followed by alkaline treatment with 27.5% removal of lignin. Roughness, surface area, and micropore volume of the biomass were crucial for the enzymatic hydrolysis. Furthermore, the ultrastructure changes observed using crystallinity, Fourier transform infrared spectroscopy, thermogravimetric analysis, and pyrolysis gas chromatography/mass spectrometry support the effects of sequential dilute acid-alkali treatment. The results provide an efficient approach to facilitate a better enzymatic hydrolysis of the poplar samples.