Abstract: Noting that most of investigations about hydrogen production were done in a small bioreactor volume (from few milliliters – 5 liter) the aim of this investigation was to observe possibility of biological hydrogen production in a continuous culture in a big lab scale with volume of 25 liter. First task of investigation was to design and set up of bioreactor for continuous biological hydrogen production. The bacterial culture used was cultivated in a batch reactor from identified bacterial mixture taken from sludge of plant wastewater treatment. Cultivation was done under anaerobic condition. Synthetic minimal medium was used as a feeding, which contained 5 gram of glucose per liter. The current investigation demonstrates the possibility of hydrogen production by dark fermentative processes in a big lab scale.