A novel biological strategy of utilizing wheat as nutrient source for hydrogen production was developed. Wheat bran was used in the solid-state fermentations of Aspergillus awamori and Aspergillus oryzae that produce amylolytic and proteolytic enzyme streams, respectively. The resulting streams were added to wheat flour to generate nutrients-rich (glucose and free amino nitrogen (FAN)) solutions. Both glucose and FAN increased with increasing of wheat flour mass ratio from 3% to 10% (w/v) and the highest glucose (43.92 g/L) and FAN (944.53 mg/L) were obtained at wheat flour mass ratio of 10%. The wheat flour hydrolyzates were then used as the feedstock for biohydrogen production by Bio-hydrogenbacterium R3. The highest cumulative hydrogen production of 4.52 L was happened at glucose concentration of 33.15 g/L. However, the maximum hydrogen yield (2.34 mol H-2/mol glucose) and hydrogen production (0.074 L H-2/g wheat flour) were obtained at glucose concentration of 16 g/L. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.