To enhance photo-fermentative hydrogen production (PFHP), biofilm reactor (BR) was employed as an ideal strategy with optimization on key factors of acetate concentration and carriers in this work. Optimal conditions for hydrogen production were acetate concentration of 4 g/L and carriers (silicon sheet) of 10 cm x 1 cm at amount of 1 piece. Biofilm formed on silicon sheet strongly improved hydrogen production compared with control reactor (CR). Cumulative hydrogen volume was enhanced about 20% from 2850 +/- 130 mL/L of CR to 3349 +/- 153 mL/L of BR and hydrogen yield was increased 20% from 2.61 +/- 0.13 mol H-2/mol acetate of CR to 3.06 +/- 0.15 mol H-2/mol acetate of BR at 4 g/L acetate. Protein and deoxyribonucleic acid (DNA) were important components to form the biofilm and they occupied 90% of extracellular polymeric substances (EPS). In particular, DNA, nearly 50% content of EPS, likely indicated a substantial contribution to biofilm formation and bacterial communication. Moreover, it suggested biofilm could regulate free cells to decline EPS secretion for improved hydrogen production. This work indicates BR could be a promising and economic strategy to enhance hydrogen production by photo-fermentation. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.