Hydrogen can be produced by fermenting sugars in a mixed bacterial culture under anaerobic conditions. The low retention rate of hydrogen producing bacteria limits the productivity of a suspended-growth reactor due to the requirement for long hydraulic resident time (HRT) to maintain adequate bacteria population. Anaerobic granular sludge was proposed as immobilized hydrogen producing bacteria to be used in the immobilized hydrogen culture after methanogenic activity of granule was eliminated by chloroform treatment. Chlorofrom treated methanogenic granules were packed into an upflow reactor and were investigated for the continuous hydrogen fermentation. Experimental results indicated that there were many factors influencing the reactor performance including initial pH of the culture medium, calcium ion addition, glucose loading concentration, and hydraulic retention time, etc. Additional formation of self-flocculated granular sludge occurred during operation at a short HRT.  SEM and TEM pictures showed that methanosaeta was totally eliminated from the granule at the pretreatment stage, while the newly produced granules, formed by rod-like bacteria, had a diameter of about 0.7 mm and a very porous structure. 16r DNA based microbial analysis showed that hydrogen producing bacteria dominate both types of granule. The packed methanogenic treated granule and additionally formed granule can lead to a high biomass concentration in the reactor which significantly increased the hydrogen production rate.