Anaerobic process of biohydrogen production was developed recently. The representative strain of hydrogen production bacteria, namely fermentative hydrogen producing bacterial strain B49，was isolated from the activated sludge which was cultured artificially by our lab. In this study, there were some different C/N ratios employed. Experiment results indicated that the concentration of carbon and nitrogen had a great effect upon hydrogen production of B49, when C/N=2.5, glucose got the highest degraded efficiency, and at a C/N ratio of 3.3, the bio-gas and hydrogen production peaked with 159mL and 37.5mL, respectively, which considered that the optimum C/N ratio on the growth and hydrogen production was 3:1. Furthermore, the mainly terminal liquid products of B49 were ethanol and acetic acid. With the increase of the total organic nitrogen, final pH value decreased gradually.