In this study, sludge characteristics of anaerobic sequencing batch reactor (ASBR) were investigated to improve and optimize the efficiency of the system converting starch into biohydrogen. The effect of stratification in settling phase on H2-producing ASBR, which results in settleable and non-settleable sludge, was observed using a batch experiment. It was concluded that specific H2 activity of decanting non-settleable sludge was higher than that of settleable sludge, which may be the reason of low yield in H2-producing ASBR. In addition, effect of settling time on settleable sludge, which is another key operational parameter, was also analysed using another set of batch experiment. Settling time of the sludge was found to be an important parameter in H2-producing ASBR. Specific H2 activity varied inversely with the duration for which settleable microorganisms were contained in settling phase. Microbial species, responsible for H2 activity in each condition, were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis.