Increasing reducing sugars(xylose and glucose) yield for bioethanol from corn stover depends strongly on optimization of pretreatment conditions. The optimum reaction conditions of two-stage liquid hot water(LHW) pretreatment based on total sugars yield were investigated. Under optimal conditions, the recovery of glucose of corn stover after two-stage LHW pretreatment and 72 h enzymatic digestion, reached 89.55%. In addition, acetic acid-rich spent liquor pretreatment and one-stage LHW pretreatment have been carried out to make comparisons with two-stage LHW treatment. Glucose yield 89.55% is superior to the recovery 83.38% using acetic acidrich spent liquor pretreatment or 80.58% using one-stage LHW pretreatment. The production of total sugars was increased by 7.8% when compared with one-stage pretreatment. Moreover, the structural features of the treated corn stover solid residues were also investigated by XRD and SEM technology in order to clarify the effects of the reaction on corn stover. The results indicated that the two-stage LHW pretreatment was an effective pretreatment method of corn stover to get most massive resource utilization, and it could be successfully applied to corn stover. Increasing reducing sugars (xylose and glucose) yield for bioethanol from corn stover depends strongly on optimization of pretreatment conditions. The optimum reaction conditions of two-stage liquid hot water (LHW) pretreatment based on total sugars yield were investigated. Under optimal conditions, the recovery of glucose of corn stover after two-stage LHW pretreatment and 72 h enzymatic digestion, reached 89.55%. In addition, acetic acid-rich spent liquor pretreatment and one-stage LHW pretreatment have been carried out to make comparisons with two-stage LHW Treatment of Glucose yield 89.55% is superior to the recovery 83.38% using acetic acidrich spent liquor pretreatment or 80.58% using one-stage LHW pretreatment. The production of total sugars was increased by 7.8% when compared with one-stage pretreatment. Moreover, the structural features of the treated corn stover solid residues were also investigated by XRD and SEM technology in order to clarify the effects of the reaction on corn The results indicated that the two-stage LHW pretreatment was an effective pretreatment method of corn stover to get the most massive resource utilization, and it could be successfully applied to corn stover.