Abstract: Abstract: Energy crisis and environmental pollution make it necessary to find an alternative energy source that is environmentally friendly and renewable. Hydrogen is generally considered as a clean and renewable energy. There are many hydrogen production methods in physical, chemical and biological ways. Hydrogen production from agricultural waste by anaerobic fermentation can not only solve the disposal of agricultural waste, but also obtain clean and efficient energy. This approach achieves the reduction of agricultural waste and resource's harmless utilization with easy operation and low cost, and it is considered to be one of the best and most promising methods of hydrogen production. In this paper, according to the characteristics of corn straw, i.e., compact structure and being difficult to directly degrade, the effect of pretreatment methods on the hydrogen production from corn straw was studied. In the first place, the corn straw was soaked in hydrochloric acid (HCl) solution of 0.2%, 0.4%, 0.6% and 0.8% at room temperature for 24 h, and then the hydrogen production experiments were taken under the warm fermentation condition (38℃) and the anaerobic activated sludge was used as the inoculum. The results showed that the cumulative hydrogen production and hydrogen production rate of corn straw increased firstly and then decreased with the increase of HCl concentration; 0.6% HCl pretreatment had the best performance: cumulative hydrogen production per unit was 87.90 mL/g, 155% more than the corn straw without pretreatment, and the hydrogen production rate was 3.05 mL/(g.h). Furthermore, different concentrations of HCl had little effect on the hydrogen concentration of gas produced by corn straw fermentation. Next, in the condition of (38±2)℃, single factor anaerobic fermentation test and orthogonal anaerobic fermentation test were carried out to investigate the effects of straw particle size, substrate concentration and initial pH value on hydrogen production process, using corn straw pretreated with 0.6% HCl as raw material. The results demonstrated that the smaller the corn straw particle size was, the more the amount of generated hydrogen was produced; a modest increase in substrate concentration could increase the hydrogen production; suitable initial pH value of fermentation was in favor of the growth and reproduction of hydrogen- producing bacteria; optimized parameters of the process were considered to be: Corn straw particle size of 150 μm, substrate concentration of 15 g/L and initial pH value of 7, and the cumulative hydrogen yield was 112.87 mL/g under the optimized parameters of the process. The main limiting factor for hydrogen production by anaerobic fermentation was corn straw particle size, followed by substrate concentration, and initial pH value of fermentation played less important role in the hydrogen production performance of corn stalk. This paper provided the reference and instruction for hydrogen production with corn straw and other agricultural organic wastes.