Abstract Forest wood biomass can be a sustainable and cost-effective feedstock for the biorefinery industries, but the rigid and compact structure of plant cell is a major barrier for production of clean energy and biochemical. In this case, the MIBK/H2O pretreatment with biphasic system was applied to treat Eucalyptus and then systematically evaluated pretreatment conditions (e.g., MIBK/H2O ratio, reaction temperature and time, NaCl concentration, and HCl dosage) on the effect of furfural yield. The resulting solid fraction obtained from the optimum pretreatment condition for furfural yield was to produce fermentable glucose by enzymatic hydrolysis. Study on enzymatic hydrolysis of the raw material and resulting solid fraction obtained by single aqueous system was also contrastively investigated. The furfural yield was 65.9% and the recovery of residue was 46.9% under an optimal reaction condition (VMIBK:VH2O = 5:5, 150 °C, 60 min, 0.3 M HCl). Meanwhile, the yield of glucose of cellulose was improved after the pretreatments with different systems and a maximum value was up to 60.2% by the MIBK/H2O pretreatment. The effective fermentable glucose production was mainly affected by the significant removal of hemicelluloses, change of CrI, and destruction of surface morphology of Eucalyptus. The MIBK/H2O pretreatment can be considered as a potential approach for efficient conversion of Eucalyptus to clean energy and biochemicals.