An ethanol-based hydrogen producing bacteria Ethanoligenens R3 was isolated from continuous stirred tank reactor used for hydrogen production in previous experiment. Batch culture experiments were operated in this study with the fermentative temperature of 35°C and substrate concentration of 10g/L. The performance of hydrogen production from glucose, brown sugar and starch by Ethanoligenens R3 was investigated. The results show that the optimal substrate used for bio-hydrogen production was glucose with the maximum hydrogen yield of 834 mlH2/L culture. Furthermore, effect of initial pH of the culture medium on hydrogen production was studied. It is demonstrated that Ethanoligenens R3 reached the maximum biogas yield of 1760 ml/L culture with the maximum total VFAs concentration of 3329.39 mg/L at the initial pH of culture medium of 5.5.