This work contributes to the improvement of biomass pretreatment technologies and shows an efficient pretreatment by glycerol -water with a good lignocellulose biomass fractionation and an enhanced enzymatic susceptibility of pretreated solid. The results show that solubilized wood fraction after glycerol treatment is composed by 15.9 and 13.2 g of lignin and xylose / 100 g of raw material in the liquid phase, respectively, whereas the solid phase was hydrolyzed by enzymes (achieving up to 90 % conversion of cellulose to glucose). So, the use of glycerol in organosolv treatment is a suitable alternative for the use of an industrial by-product and the structural modification of biomass for second generation bioethanol production