Abstract: To study the pure bacteria on producing hydrogen, one pure bacterium XN12 was isolated from active sludge of wastewater plant. Based on the sequencing of 16 S rDNA and DnaJ gene and cellular morphology as well as biochemical reactions, the isolates were identified as Bacillus cereus. In the condition of glucose as the substrate and with different operational pH values, the characterization on hydrogen yielding with the pure bacterium was studied.Resultsshowed that for every mol glucose, optimum condition for maximizing H₂ production was initial pH of 6. 9, in which the maximum H₂ production was 1. 6 mol H₂. According to the metabolic production of the reactions, the strain was butyric acid fermentation type and produced hydrogen through pyruvate metabolism pathway.