Expanded granular sludge bed (EGSB) reactor and bioaugmentation were employed to investigate biohydrogen production with molasses wastewater. The start-up experiments consisted of two stages. In the first stage (0～24d) seeded with activated sludge, the butyric acid type-fermentation formed when the initial expanding rate, organic loading rate (OLR), the initial redox potential (ORP) and hydraulic retention time (HRT) were 10%, 10.0 kg COD/(m3·d), - 215 mV and 6.7 h, respectively. At the beginning of the second stage on day 25, the novel hydrogen-producing fermentative bacterial strain B49 (AF481148 in EMBL) were inoculated into the reactor under the condition of OLR 16. 0 kg COD/(m3·d), ORP and HRT about - 139 mV and 6.7 h, respectively, and then the reaction system transformed to ethanol-type fermentation gradually with the increase in OLR. When OLR, ORP and HRT were about 94.3 kg COD/(m3·d), -250 mV and 1.7 h, respectively, the system achieved the maximum hydrogen-producing rate of 282.6 mL H2/L reactor· h and hydrogen percentage of 51%～53% in the biogas.