Abstract: In recent years, hydrogen as eco-friendly clean energy has received the widespread attention. In numerous hydrogen production methods, anaerobic fermentation bio-hydrogen production has become a research hotspot in the field of renewable energy because it can utilize biomass raw materials effectively, reduce the agricultural waste pollution to the environment. The straw biomass is the main agricultural waste of crop production in China, which can be used to take direct or prepare molding fuel combustion and convert into biogas for comprehensive utilization generally. Cellulose content in maize straw (about 36%) is higher than that in wheat、soybean and sorghum straws, the research of cellulose energy efficient transformation in maize straw has important scientific significance and practical value. In this study, anaerobic fermentation experiments were performed for bio-hydrogen production from enzymatic hydrolyzate of corn stalk powder (<0.088 mm) using heat pretreated activated sludge as fermentation microorganism, choosing cumulative amount of hydrogen production as main experiment parameter，the influences of different factors on anaerobic fermentation bio-hydrogen production of corn stalk’s enzymolysis were studied based on Box-Behnken model of response surface method. The significance of interactions between different factors during the anaerobic bio-hydrogen production process was examined and the anaerobic fermentation bio-hydrogen production process of corn stalk’s enzymolysis was optimized. The results showed that temperature and enzymatic hydrolysate concentration were the factors that mostly impacted on the corn stalk anaerobic fermentation bio-hydrogen production process comparing with initial pH value. The interaction of two factors on the effect of hydrogen yield were all significant (P＜0.05). Box-Behnken model was used to obtain the optimal conditions of hydrogen production. The optimal conditions were temperature of 38.32℃, the initial pH value of 4.93, and the enzymatic hydrolysate concentration of 20.70 mg/mL. Under such condition, the maximum hydrogen yield was 685.59 mL and the maximum hydrogen production rate was 57.13 mL/g straw. The model was validated through experiment, the actual maximum hydrogen yield can reach 659.24 mL and the hydrogen production rate was 54.94 mL/g straw, with the prediction error of 3.84%, which proved that this model had a good fitness.