<P><B>Abstract</B></P> <P>Effect of substrate concentration on photo-fermentative hydrogen production was studied with a self-designed 4m<SUP>3</SUP> pilot-scale baffled photo-fermentative hydrogen production reactor (BPHR). The relationships between parameters, such as hydrogen production rate (HPR, mol H<SUB>2</SUB>/m<SUP>3</SUP>/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±8.83mol/m<SUP>3</SUP>/d was achieved in chamber #3 at a substrate concentration of 20g/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 20g/L/d, then decreased when organic loading rate was further increased to 25g/L/d. A maximum HPR of 148.65±4.19mol/m<SUP>3</SUP>/d was obtained when organic loading rate was maintained at 20g/L/d during continuous bio-hydrogen production.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Photo-fermentative H<SUB>2</SUB> production using sunlight as light source was operated. </LI> <LI> Optimal influent substrate concentration was found to be 20g/L. </LI> <LI> A hydrogen production rate (HPR) of 202.64±8.83mol/m<SUP>3</SUP>/d was achieved. </LI> <LI> Substrate concentration significantly affected HPR and broth characteristics. </LI> <LI> Optimal organic loading ratio for continuous hydrogen production was 20g/L/d. </LI> </UL> </P>