Effect of substrate concentration on photo-fermentative hydrogen production was studied with a self-designed 4 m(3) pilot-scale baffled photo-fermentative hydrogen production reactor (BPHR). The relationships between parameters, such as hydrogen production rate (HPR, mol H-2/m(3)/d), hydrogen concentration, pH value, oxidation-reduction potential, biomass concentration (volatile suspended solids, VSS) and reducing sugar concentration, during the photo-fermentative hydrogen production process were investigated. The highest HPR of 202.64 +/- 8.83 mol/m(3)/d was achieved in chamber # 3 at a substrate concentration of 20 g/L. Hydrogen contents were in the range of 42.19 +/- 0.94%-49.71 +/- 0.27%. HPR increased when organic loading rate was increased from 3.3 to 20 g/L/d, then decreased when organic loading rate was further increased to 25 g/L/d. A maximum HPR of 148.65 +/- 4.19 mol/m(3)/d was obtained when organic loading rate was maintained at 20 g/L/d during continuous bio-hydrogen production.