Biohydrogen production by anaerobic sludge was studied by using wastewater as substrate in batch process. Hydrogen production potential of different substrate species, as well as the composition of fermentation product in liquid phase, was studied in the batch culture experiments. The hydrogen production and bacterial growth dynamics were also analyzed. The experiment results demonstrated that the optimal substrate was glucose for hydrogen production. It could be obtained maximum cumulative hydrogen production 67.21 L/mol, hydrogen yield 3.23 mol/mol and hydrogen content 49.52%. The butyrate and acetate were main liquid end products, occupied 26.76%-40.49% of acetate, 37.60%-58.07% of butyrate, implying that it is butyrate-type fermentation. Butyrate/acetate could be used as the indicator for evaluating the effectiveness of H2 production, with that the higher butyrate/acetate, the higher the hydrogen yield. ORP was less than -300 mV during fermentation indicating the experiment was anaerobic. A modified Gompertz model can adequately describe the H2 production and bacterial growth.