Abstract: Increasing demand of energy due to industrialization has resulted into the shortfall of non renewable energy sources. Also the over consumption has raised many serious environmental issues, all these problem has resulted for the search of some alternative renewable energy sources which is eco-friendly and biohydrogen fulfils all these requirements. In the present study rice mill effluent was taken as a substrate and Klebsiella oxytoca ATCC 13182 strain was used for the fermentative production of biohydrogen. Hydrogen production was carried out at 40°C temperature and at 6 pH after optimization. Its quantity was measured by water displacement method. Immobilized bacteria were used for the process. Bacteria were immobilized in different concentrations of sodium alginate containing 0.3% activated charcoal. It was found that at concentration of 2 and 3% of sodium alginate 50.33±0.88 and 52.67±1.45 mL productions were observed, respectively with no significant difference. Therefore, effect of HRT (Hydraulic Retention Time) was studied at these 2 concentrations of sodium alginate. The rate of biohydrogen production was found better at concentration of 2%. Effect of metal ions, iron and cobalt was studied at different concentrations and it was found that maximum of 55.00±0.57 mL of hydrogen was produced at 80 mg L-1 concentration of FeSO4 and cobalt nitrate at a concentration of 80 mg L-1 gave maximum production of 58.33±0.88 mL. Different physicochemical parameters of waste water was analyzed before and after fermentation process.