Abstract: Abstract: Along with the rapid development of society and unceasing improvement of human living standard, the consumption of fossil fuels is rising continuously, which has resulted in several problems such as energy shortage and environmental pollution. Therefore, the research and exploitation of renewable energy become extremely urgent. Hydrogen is a promising alternative energy, and can be produced through different methods. Considering the complementarity between different hydrogen-production microorganisms, dark-photo integrated bio-hydrogen production was regarded as a beneficial way to improve the utilization rate of substrate and the potential of hydrogen production. Dark-photo integrated bio-hydrogen production could combine the characteristics of various microbes, and exert their advantages. Moreover, enzymatic hydrolysis of complex substrate, VFAs production, VFAs consumption, and hydrogen production could synchronously take place, so it could realize the reuse of byproducts (VFAs) and avoid the accumulation of liquid byproducts. In this study, enzymatic hydrolyzate of corn stover was taken as substrate, Enterobacter aerogenes (AS1.489) and photosynthetic bacteria (HAU-M1) were selected as hydrogen-production microorganisms, and simultaneous saccharification fermentative method was adopted to study the process of dark-photo integrated bio-hydrogen production by mixed cultivation of HAU-M1 and Enterobacter aerogenes. Cumulative hydrogen yield was taken as key reference to optimize the process parameters of bio-hydrogen production. The single factor experiments were adopted to select the optimal lever and analyze the effects of substrate concentration, initial pH value, light intensity and fermentation temperature on the integrated bio-hydrogen production. On the basis of single factor experiments, orthogonal experimental design was also adopted to further optimize the bio-hydrogen production process parameters and evaluate the significance of influencing factors. The results of orthogonal range analysis showed that primary and secondary order of the influence of various process parameters on integrated bio-hydrogen production was: fermentation temperature＞initial pH value＞substrate concentration＞light intensity. The analysis of variance showed that fermentation temperature and initial pH value were the most significant factors affecting integrated bio-hydrogen production and produced the most significant influence on the process of integrated bio-hydrogen production by mixed cultivation of photosynthetic bacteria and Enterobacter aerogenes. The optimum process parameters were: substrate concentration of 35g/L, initial pH value of 6.5, light intensity of 3500lx, and fermentation temperature of 30 ℃. The validation experiments under these conditions were performed, and the cumulative hydrogen yield of 332.6 mL for 72 h and the capacity of unit hydrogen production of 47.5 mL/g corn stover were obtained. The optimal process parameters for bio-hydrogen production provide a scientific reference for the further research on integrated bio-hydrogen production by mixed cultivation of dark-fermentative and photo-fermentative bacteria from straw biomass.