AbstractThree-step extraction of lignin fractions from ball-milled sugarcane bagasse (SCB) was studied with 96% dioxane, 50% dioxane, and 80% dioxane containing 1% NaOH at boiling temperature followed by purification to remove hemicelluloses. The total yields of hemicelluloses and lignin were 15.8% and 7.2% based on dried SCB, respectively. In the first step, 5.1% lignin (70.8% of the total extracted lignin) was isolated with 96% dioxane, which is higher than the traditional MWL procedure (3.3% lignin). In the second step, 10.4% hemicelluloses (65.8% of the total extracted hemicelluloses) were obtained with 50% dioxane. The obtained lignin fractions were characterized with FT-IR, 31P-NMR, and 2D 1H-13C HSQC. The results suggested that the ‘core’ of the lignin structure did not change dramatically during the sequential neutral and alkaline dioxane treatment processes. The contents of phenolic hydroxyl groups in the three lignin fractions based on 31P-NMR analysis were 1.66, 4.46, and 9.42 mmol/g lignin, respectively, higher than those from wood. The results also indicated that the lignin fractions obtained from SCB contained some amount of p-coumaric acid and ferulic acid, significantly different from those extracted from softwood and hardwood.