In this work, the synergistic effects of L-cysteine and biofilm on the performance of reactor were investigated in the photo-fermentative biofilm reactor. Both L-cysteine and biofilm could enhance the performance of reactor for producing hydrogen. The optimal L-cysteine concentrations in the biofilm reactor (BR) and control reactor (CR, reactor with no biofilm) were 0.5 g/L and 0.75 g/L, respectively. In addition, the maximum cumulative hydrogen volume (3500 +/- 170 mL/L), substrate conversion efficiency (79.4 +/- 4.0%) and hydrogen yield 867 +/- 43 mL/g (acetate) were obtained in the BR at L-cysteine concentration of 0.5 g/L. It is intriguing that extracellular DNA (eDNA) occupied 50% of EPS, which could play a key role in hydrogen production. Biofilm could decrease EPS concentration of cells in the BR at each L-cysteine concentration to divert more energy into hydrogen. The moderate L-cysteine concentration could improve bacterial growth and regulate the EPS to promote the biofilm formation and enhance hydrogen production. Thus, the regulation of L-cysteine concentration will be a practical method to enhance hydrogen production by the biofilm reactor. (C) 2019 Elsevier Ltd. All rights reserved.