Abstract: With the steam-explored corn stover as the substrate, the effects of multi-stage hydrolysis and the reuse of β-glucosidases via ultrafiltration technique on enzymatic hydrolysis yield were investigated. The analysis results indicated that the removal of cellobiose and glucoseat each stage was benefit to improve the cellulase activities, the hydrolysis efficiency was enhanced and less hydrolysis time was obtained. Higher hydrolysis yield (70.16%) was achieved as three-stage (6+6+12) h hydrolysis was used in 24 h in contrast to a 62.84% yield in 72h for one-stage hydrolysis. The steam-explored corn stover was composed by 23% crystalline region and 77% amorphous region. Two diffraction peaks of substrate at diffraction angle of 16° and 22° were prominent after the three-stage (6+6+12) h hydrolysis, and the diffraction intensity increased. The ultrafiltration technique with the 30 ku membrane was employed to recover the β-glucosidase. The glucose yield during the first round of hydrolysis was 92.71%, and the β-glucosidase recovery rate was 98.87%. During the 8th round of hydrolysis, the glucose yield and the β-glucosidase recovery rate were 90.87% and 95.25%, respectively.