To achieve stable and efficient photo-fermentative hydrogen production, this work investigated photo-fermentative hydrogen production by forming biofilm on the surface of carrier in the biofilm reactor (BR). Results showed the hydrogen production performance was greatly improved by formed biofilm. The time of hydrogen production and efficiency of substrate utilization were enhanced obviously compared to the control reactor (CR). When the CR was used, hydrogen production stopped at 7th day and maximum cumulative hydrogen volume and hydrogen yield were 1730 +/- 87 mL/L and 1.44 +/- 0.07 mol H-2/mol acetate, respectively. However, in the BR hydrogen production volume of 3028 150 mL/L and hydrogen yield of 2.52 +/- 0.13 mol H-2/mol acetate were obtained, which were enhanced about 75% compared to that of the CR. The time of hydrogen production extended from 7 days of CR to 12 days of BR and the substrate conversion efficiency increased from 36% of CR to 63% of BR. It was worth noting at 8th day that substrate was almost utilized completely but hydrogen production still lasted for 4 days. This suggested that the formation of biofilm in BR was favorable to continuous hydrogen production and substrate utilization with high efficiency. Results demonstrated the BR can get a more stable and consistent operating process and it was a proper and potential way to produce hydrogen by photo-fermentative bacteria (PFB). (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.