Hydrogenase is one of the vital rate-limiting enzymes in biohydrogen production metabolism. The activity of hydrogenase impacts the speed of hydrogen production. In the present study, the novel high efficient H-2-producing bacterium Ethanoligenens harbinense YUAN-3 was selected for characterization of the relationship between hydrogenase activity and its hydrogen production performance. The effects of the ecological factors of temperature, pH, C/N ratio Mg2+, and Fe2+ concentration on hydrogen production and the activity of hydrogenase were investigated. The activity of hydrogenase decreased quickly after reaching a maximum rate of 2.18 mol H-2 molGlucose(-1) (H-2 yield of 1942.5 mL L-1) along with the fermentation process. The highest hydrogenase activity of strain E. harbinense YUAN-3 was optimized under conditions of 35 degrees C, pH 4.5, and the optimal C/N ratio of 22.4. It was also found that the optimal concentration of 300 mg L-1 MgCl2 center dot 6H(2)O and 300 mg L-1 FeSO4 center dot 7H(2)O were playing key roles in hydrogen production. The highest hydrogen production was improved to 2.62 mol H-2 molGlucose(-1) (H-2 yield of 2138.5 mL(-1) with the maximum activity of the hydrogenase 0.352 U mg(-1) protein. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.