Improving bio-hydrogen-producing capacity is the key to realize industrialization for the H2-producing technology. One of the most effective approaches is to investigate the factors that affect the H2-producing capacity. Different glucose concentrations and pH were designed in anaerobic Clostridium R33 sp.nov. cultures. The hydrogen production varies according to glucose concentration and pH, respectively. It is founded the optimal glucose concentration is 12.5 g/L and the optimal pH is about 4.0.