Abstract Humic acid (HA) containing in the inoculated waste activated sludge (WAS) would be released into the fermentation liquid during dark fermentative hydrogen production. Nevertheless, the influence of HA on the photofermentative hydrogen production from volatile fatty acids (VFAs) left in the dark fermentation liquid by photosynthetic bacteria (PSB) had not been investigated. This study measured the effects of sludge humic acid (SHA) and AQS (anthraquinone-2-sulfonic acid, model humic acid) on the photofermentative hydrogen production from VFAs. Results showed that the photofermentative hydrogen production was reduced by 12.0% and 35.4% in synthetic wastewater with 100 mg/L of the added SHA or AQS, respectively. Mechanistic studies showed that high concentrations of SHA and AQS inhibited the activity of nitrogenase and development of PSB biomass, as well as damaging the cell membranes and causing significant death of PSB, leading ultimately to a significant decrease of photofermentative hydrogen production. Moreover, fluorescence spectra showed that SHA had a small molecular weight and a low degree of humification compared to that of AQS, which caused more negatively influence of AQS on photo hydrogen generation from VFAs. Finally, the feasibility of removing SHA from anaerobic dark fermentation liquid of wastewater to improve photofermentative hydrogen production was testified.