Three fixed-bed materials including chlorinated polyethylene (CPE), loofah sponge (LS) and porous nylon (PN) were investigated for enhancing bio-hydrogen production in anaerobic digestion. In the batch operation, the bioreactor fixed with CPE achieved the highest hydrogen production of 676.4 mL/L (5 g-glucose/L). The SEM observation revealed that CPE bioreactor immobilized the highest density of microorganisms, facilitating the conversion of hydrogen through favorable butyrate pathway as well as maintaining stable pH value at optimal range. Among the three fixed-bed materials, CPE was obtained the optimum bedding material due to its roughness surface and high porosity. Additionally, the bioreactor fixed with CPE remained high hydrogen production and carbohydrate degradation under high concentration of glucose (35 g/L), whereas the no fixed-bed reactor (Control) showed negligible hydrogen production. The results indicated that CPE bioreactor could be tolerant to environmental perturbation, being more stable in operation, promoting a higher biological activity due to the higher cell density. Furthermore, the CPE bioreactor proved to be high-efficient and stable for hydrogen production with 32 days long-term operation. This is the first work revealing that the CPE bioreactor is promising for improving bio-hydrogen production.