Abstract: For developing new pretreatment methods of fuel ethanol production, hemicellulose separation two-step pretreatment method and lignin separation two-step pretreatment method were investigated using switchgrass and corn stover. In the hemicellulose separation two-step pretreatment, a cellulose digestibility of 89% was obtained (Step 1 with the condition of 2%wt H2O2, 50oC and 16 h; Step 2 with the condition of 121oC and 30 min), while in the lignin separation two-step pretreatment, the highest cellulose digestibility of corn stover was 83% (Step 1 with the condition of 1%wt NaOH, 70%wt ethanol solution, 80oC and 2 h; Step 2 with the condition of 135oC and 30 min). SEM micrographs showed that the surface of biomass had been altered and the accessibility of enzymes had been improved. Hemicellulose and lignin separated from the feedstock could be recovered for the production of value-added products. The results indicate that these two pretreatment methods have a promising future, and are very helpful for the industrialization of fuel ethanol production.