AbstractReducing sugars contained in agricultural residue hydrolyzates can potentially be utilized in microbial fermentations to produce biofuels and biogas. Different types of agricultural residues were employed for photo-fermentative bio-hydrogen production, and the cumulative hydrogen production data fit well to the Modified Gompertz Model. Corncob was determined to have the highest reducing sugar yield and cumulative hydrogen production (12.64 mg mL-1 enzymatic hydrolysate, 228.94 mmol L-1) and maximum hydrogen production rate (5.9677 mmol L-1 h-1). Enzyme reuse was investigated by single factor experiment design to reduce the cost of bio-hydrogen production. Taking reducing sugar yield and activity recovery efficiency as reference, substrate re-adsorption method at different temperature and time, then enzyme immobilization method at different load and pH were investigated in the process of enzymatic hydrolysis. The efficiency of enzyme utilization was enhanced via substrate re-adsorption and enzyme immobilization methods, which resulted in a 4-fold increase in recycling efficiency. The optimal enzyme reuse condition by substrate re-adsorption was a re-adsorption time of 90 min at a temperature of 15 °C, while the optimal condition by enzyme immobilization method was a pH of 4.8 and immobilized enzyme load of 400 mg.