Summary There are many factors affecting the dark fermentative hydrogen production. The interaction of these factors, that is, their combined effects, should be investigated for better design of the systems with stable and higher hydrogen yields. This study aimed to investigate the combined effects of initial substrate, pH, and biomass (or initial substrate to biomass) values on hydrogen production from sucrose and sugar‐beet molasses. Therefore, optimum initial chemical oxygen demand (COD), pH, and volatile suspended solids (VSS) or initial substrate to biomass (VSS) ratio (S/Xo) values leading to the highest dark fermentative hydrogen production were investigated in batch reactors. An experimental design approach (response surface methodology) was used. Results revealed that when sucrose was the substrate, maximum hydrogen production yield (HY) of 2.3 mol H2/mol sucroseadded was obtained at initial pH of 7 and COD of 10 g/L. Initial S/Xo values studied (4–20 g COD/g VSS) had no effect on HY, while the initial pH was found as the parameter mostly affecting both HY and hydrogen production rate (HPR). When substrate was molasses, initial COD concentration was the only variable affecting HY and HPR. Maximum of both was achieved at 10 g/L initial COD. Initial VSS values studied (2.5–7.5 g/L) had no effect on HPR and HY. This study also indicated that molasses leads to homoacetogenesis for potentially containing intrinsic microorganism and/or natural constituents; thus, sucrose is more advantageous for hydrogen production via fermentation. Homoacetogenesis should be prevented for effective optimization via response surface methodology, if substrate is a natural carbon source potential to have intrinsic microorganisms. Copyright © 2017 John Wiley & Sons, Ltd.