Experiments were carried out to investigate anaerobic fermentation hydrogen production process and its metabolic regulation ways with taking Enterobacter aerogenesenes as pure strains and using glucose as substrate. The experiments were conducted to study at different temperatures (25℃,30℃,35℃,38℃), initial pH values (3.5,4.5,5.5,6.5), mass ratio of glucoses (G) and protease digestion (2,3,6). The results showed that Enterobacter aerogenesenes had a certain acid-resisting in the pH value range of 7.0 to 4.5. Moreover, fermentative hydrogen production cycles of Enterobacter aerogenes was very variable (between 68 h to 156 h) under different technical conditions. The optimum conditions of the shortest fermentative hydrogen production cycle of 68 h were as follows:the temperature was 35℃,the pH value was 6.5, meanwhile, the carbon-nitrogen ratio was 3. Based on these conditions, hydrogen production rate and hydrogen conversion rate could reach to the value of 261.5 mL/g and 2.1 mol/mol.