The experiment took a continuous stirred tank reactor as a reacting device, which based on molasses wastewater and brown sugar wastewater as the fermentation substrate. We studied the hydrogen production capacity with the two different substrates under the anaerobic fermentation. The results showed that the maximum hydrogen production yield were 0.99 L/d (molasses as the substrate) and 1.95L/d (brown sugar as the substrate), respectively, with the hydraulic retention time (HRT) of 6.2 h, temperature of (35 ± 1) °C and the reactor substrate concentration of 3000 mg COD/L. At the same time, the liquid end components were changed in a large scope. During the first 28 days, acetic acid and propionic acid were the main components, when molasses was replaced by brown sugar as the substrate, the percentages of propionic acid and butyrate acid decreased significantly from 25.7%, 15.4% to 11.8%, 9.47%, respectively. And the fermentation type was transformed from the mixed-acid-type fermentation to the more stable ethanol-type fermentation.