The pH and hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR) were varied to optimize the conversion of carbohydrate-rich synthetic wastewater into bio-hydrogen. A full factorial design using evolutionary operation (EVOP) was used to determine the effect of the factors and to find the optimum condition of each factor required for high hydrogen production rate. Experimental results from 20 runs indicate that a maximum hydrogen production rate of 4,460-5,540 mL/L/day under the volumetric organic loading rate (VOLR) of 75 g-COD/L/day obtained at an observed design point of HRT = 8 h and pH = 5.7. The hydrogen production rate was strongly dependent on the HRT, and the effect was statistically significant (P < 0.05). However, no significant effect (P > 0.05) was found for the pH on the hydrogen production rate. When the ASBR conditions were set for a maximum hydrogen production rate, the hydrogen production yield and specific hydrogen production rate were 60-74 mL/g-COD and 330-360 mL/g-VSS/day, respectively. The hydrogen composition was 43-51%, and no methanogenesis was observed. Acetate, propionate, butyrate, valerate, caproate, and ethanol were major liquid intermediate metabolites during runs of this ASBR. The dominant fermentative types were butyrate-acetate or ethanol-acetate, representing the typical anaerobic pathway of Clostridium species. This hydrogen-producing ASBR had a higher hydrogen production rate, compared with that produced using continuous-flow stirred tank reactors (CSTRs). This study suggests that the hydrogen-producing ASBR is a promising bio-system for prolonged and stable hydrogen production.