Eucalyptus was fractionated with mild alkaline process, and the obtained cellulose fraction was pretreated with various ionic liquids (ILs) to enhance the enzymatic saccharification. The results showed that the ILs used was efficient for the hydrolysis of cellulose, with the maximum total reducing sugars (TRS) yield over 80% at 50 degrees C. The regenerated cellulose substrate exhibited a significant improvement about 4.4-6.4 folds enhancement on saccharification rate during the first 4 h reaction. The crystallinity index (Crl) of cellulose via 1-ally-3-methylimidazolium ([AMIM]Cl) pretreatment was significantly decreased from 70.2% to 31.2%, resulting in structural change from cellulose 1 to cellulose H, which enabled the cellulase enzymes easier access to hydrolyze cellulose. However, 1-butyl-3-methylimidazolium acesulfamate ([BMIM]Ace) pretreatment had no large effect on the Crl although a high conversion yield in glucose was obtained. The surface morphologies of the regenerated substrate which was pretreated via 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) showed more porous and incompact network of cellulose when compared with the untreated substrate. This result indicated a better accessibility by cellulases to the cellulose surface. Besides, a certain amount of catalysts such as MgCl2 and H2SO4 could improve the rate of enzymatic saccharification. (C) 2013 Elsevier Ltd. All rights reserved.