Effectively overcoming recalcitrant lignocellulose is the largest obstacles to cellulosic ethanol production because natural biomass composite has a limited substrate accessibility to cellulase or catalysts. Quantitative determination of cellulose accessibility to cellulase (CAC) was established based on adsorption of the non-hydrolytic protein TGC containing a cellulose binding domain (CBM) and green fluorescence protein (GFP) (Hong et al. 2007. Langmuir 23: 12535). Here we proposed a new approach to determine the surface area of cellulose and non-cellulose (lignin-rich) fractions of pretreated corn stover, switchgrass, and common reed by cellulose-solvent-based lignocellulose fractionation (CSLF). Quantitative data suggest that CSLF can break lignocellulosic structure efficiently than dilute acid, resulting in much higher surface areas for cellulose and lignin fractionation than does dilute acid pretreatment. High cellulose surface area results in faster hydrolysis rates and high cellulose digestibility. Our results infer that increasing cellulose accessibility is more important than decreasing lignin content or lignin surface area.