The biohydrogen production is promising for the alternative green hydrogen. The two-stage of the dark and photo-fermentation system increases the hydrogen production yield. In this study, the experiments carried out the cell immobilizations on dark and photo bacteria; and liquid suspended photo bacteria for those two-stage batch systems. The optimized parameters obtained, such as materials for immobilized cells, different substrates, and the substrate concentrations for hydrogen fermentation.The experiment results showed pure substrate glucose could produce a higher amount of hydrogen in the photo immobilized cells, the hydrogen concentration was up to about 40%, the utilization rate of glucose was as high as 99.41%, the accumulated hydrogen volume was 169.13 mL, and the hydrogen yield, HY, was 0.63 mol H2/mol glucose. In addition, the activated immobilized cells are used for two-stage fermentation to generate hydrogen, HY 1.75 mol H2/mol substrate. The two-stage batch fermentations for producing hydrogen, used the Gompertz equation model to obtain the cumulative hydrogen volume and hydrogen production rate (HPR). When the ratio of dark immobilized cells (DIMC) to photo suspended liquids (PSL) is 1.0:5.0, the hydrogen production shows well performance.Moreover, an advanced molecular biological technique, next-generation sequencing (NGS) with ultra-high-throughput DNA output, was applied to perform microbial communities in a different stage of hydrogen fermentation. The NGS help to understand the detailed microbial community change in such fermentation processes and provide better biological efficacy via clarifying the role of microorganisms and their interactions. After the experiments, the Clostridia increased to 28%, and the Enterobacter also increased to 15%, therefore the Rhodobacter and Bosea were reduced from 55% to 17%. Understand the detailed composition of microbial populations in stages of such bio-system will provide the useful information via clarify the biological role of microorganisms and its interactions.