To investigate the characteristics of hydrogen production by a novel fermentative hydrogen-producing bacterial strain B49 (AF481148 in EMBL), batch experiments are conducted under different conditions. Hydrogen production has a correlation with cell growth and the consumption of glucose and soluble protein. The optimum pH for cell growth is 4.5±0.15. At acidic pH 4.0±0.15, the bacteria has the maximum accumulated hydrogen volume of 2382 ml/L culture and the maximum hydrogen evolution rate of 339.9 ml/L culture*h with 1% glucose. The optimum temperature for cell growth and hydrogen production is 35℃. In addition, fermentative hydrogen-producing bacterial strain B49 can generate hydrogen from the decomposition of other organic substrates such as wheat, soybean, corn, and potato. Moreover, it can also produce hydrogen from molasses wastewater and brewage wastewater, and hydrogen yields are 137.9 ml H2/g COD and 49.9 ml H2/g COD, respectively.