The objective of this work is to investigate the fermentation capacity and metabolic characteristics of a novel strain of bacteria B49 isolated from anaerobic activated sludge. The examination was conducted in batch culture at 35 ℃. The results showed that the carbon flow gave priority to the production of ethanol, and yield of ethanol is always greater than that of acetic acid. The hydrogen and ethanol occurred simultaneously. The exponential phase of the B49s cell growth was from 12 to 22 h. Evolution of hydrogen appeared to start after the exponential phase of cell growth and reach maximum production at the early stationary phase. The rate of hydrogen production reached a maximum of 16.8 mL/h, and the percentage of hydrogen gas in the headspace of serum bottle obtained a maximum of 41% at 22 h. The B49 was able to grow using molasses as substrate for cell growth. When the molasses was used as substrate, maximum yield of hydrogen was obtained 2460 mL/L culture at 2% (V/V) of molasses. The hydrogen yield was increased to 3060 mL/L culture after addition of 0.5 g/L of yeast extract in the molasses medium and the yield of hydrogen was increased by 24.4%.