Kshipra is holy River that originate from the Kakri-Badi (indore) of Madhya Pradesh and flows across Ujjain city (23º18’N and 75º77’E). River flows across Malwa plateau to join river Chambal which later joins Gangetic system. In the present study, two study sites with high anthropogenic activities were selected on the banks of river Kshipra; they include Ramghat, and Mangalnath. Hydrogen is view as a very clean energy source, since its combustion release mainly dihydrogen monoxide as a reaction product additionally it has advantage of having the highest energy density when compared to any other fuel. This research deals with the fermentative production of biohydrogen gas and removal of pollution load from Khsipra River wastewater. In this study various parameters like contact time, pH value, reactor operating temperature are considered. An anaerobic batch reactor along with at magnetic stirrer having working volume of 1.5 litres was constructed and operated for 12 days. The temperature of the CSTR was regulated at atmospheric temperature. Biohydrogen gas obtained after 12 days about 92 ml at pH value 6.0-5.6, atmospheric temperature 26±2 ºC and colour of the substrate was Dutch white.