Ethanoligenens sp. R3 isolated from anaerobic sludge in a continuous stirred tank reactor was investigated for its characteristics of fermentative hydrogen production in this study. Effects of initial pH value, fermentation temperature and proportion of carbon/nitrogen source on hydrogen production were investigated in batch experiments. The typical substrate for hydrogen production of glucose and peptone were used as the carbon source and nitrogen source, respectively. The results show that the maximum hydrogen production yield of 834 mlH2/L culture was obtained with initial pH value of 5.5 and fermentation temperature at 35°C. In addition, the maximum average hydrogen production rate of 10.87mmolH2/g cell dry weighth was achieved at the carbon/nitrogen source ratio of 3.3. The degradation efficiency of the glucose used as the carbon source ranged from 91.5% to 95.43% during the conversion of glucose to hydrogen by bacteria Ethanoligenens sp. R3.