Abstract: To investigate the effects of formaldehyde dosage on improving enzymatic hydrolysis efficiency and ethanol fermentation yield of sugarcane bagasse, the sequential NaOH and hydroxymethylation pretreatment (AHP) were used to treat sugarcane bagasse (SCB) with different proportion of formaldehyde by hydroxymethylation reaction. The results showed that when SCB was treated by AHP with substrate and formaldehyde ratio of 1:1.5 for 2 h, the delignification rate was 55.68% and the mass fraction of cellulose was 67.20%.Under the cellulase dosage of 20 FPIU/g(based on the mass of glucan), the 72 h glucose yield was 89.61%, which was 8.86 times higher than that of untreated SCB (9.09%). After 24 h fermentation, the ethanol concentration increased from 10.93 g/L of AP-SCB to 12.96 g/L of AHP-SCB-1.5.The structural features of the pretreated sugarcane bagasse were measured by FT-IR, and the hydrophobicity and cellulase accessibility were studied. The hydrophobicity (0.127 L/g) and enzymatic accessibility (505.73 mg/g) of AHP-SCB-1.5 were 64.64% and 1.11 times of AP-SCB, respectively. The results showed that alkali and hydroxymethylation had a synergistic effect on pretreatment process, which could improve the hydrophilicity and cellulase accessibility of materials.