Using response surface method, 20 runs of experiments were carried out to investigate the effects of initial pH variation (5-8), temperature (25-40 degrees C), and glucose concentration (4-12 g/l) on biohydrogen production in dark fermentation method by Clostridium acetobutylicum (PTCC 1492). Results show that the raise of temperature to 37 degrees C leads to increase of hydrogen production volume and yield. It is also observed that while the increase of pH to 6.5 increases hydrogen production, further increment of pH declines volume and yield. Finally, it is found that increase of glucose concentration from 8g/l leads to decrease of yield and increase of production volume.