A full factorial experimental design was employed in this experiments for learning the effects ofpH and hydraulic retention time (HRT) on hydrogen production in dark fermentation by a mixed culture of fermentative bacteria using a laboratory-scale anaerobic sequencing batch reactor (ASBR). Five pH values ranging from pH 4.7 to 5.9 at an increment of 0.3 and four HRTs ranging from 12 to 24 hours at an increment of 4 hours were tested. The results showed that at37±1°C, both pH and HRT had a strong influence on the reactor performance including gas volume, gas composition, reactor stabilization, microbial growth, and volatile fatty acids (VFAs) and alcohol distribution. The optimal condition for hydrogen production was found at HRT 16h and pH 5.0, under which the biogas comprised 35.8 ±1.6% of hydrogen with a production rate of 9.8 ± 2.2L-H2/day, a specific yield of 0.83 L-H2/ (g biomass), and a yield of 1.63 ± 0.06 L-H2/L liquid swine manure, which demonstrated a significant and encouraging product to substrate ratio of 1.63. The highest butyrate to acetate ratio, the lowest propionate concentration, as well as a satisfying stabilization and biomass concentration were all observed at the optimal condition in the anaerobic sequencing batch reactor system.