Using renewable biofuels is an effective way to solve the environmental and economic problems caused due to the over use of fossil fuels. Since the industrial effluent contains high chemical oxygen demand (COD) concentration, it could be sufficiently managed and converted to biofuels such as biohydrogen and biogas via biological processes. In this study biological production of biohydrogen and biogas from paper mill effluent as feedstock in a two stage sequencing system was evaluated. Different pretraetment conditions were tested in order to obtain a mixed microbial inoculum for dark hydrogen fermentation which heat-shock pretreatment of anaerobic sludge at 90 ?C for 15 min was chosen for further experiments. The effect of substrate hydrolysis was investigated using different acidic, thermal- acidic, and alkaline hydrolysis. Alkaline hydrolysis led to the maximum hydrogen yield of 31/1 mL H 2 /g-COD initial which gives 3% improvement of yield comared to control sample. Therefore, paper mill effluent was used without hydrolysis for further experiments. The influence of substrate concentrations, initial pH, and fermentation temperature on biohydrogen production in dark fermentation was investigated. First, the impact of initial pH (5, 6 and 7) on the efficiency of hydrogen fermentation at different concentrations of substrate (3, 5 and 7 g-COD/L) was investigated, which the maximum hydrogen yield was obtained at initial pH of 5 at all amount of substrate concentration. After that, the effect of substrate concentration (3, 5, 7, 10 and 15 g-COD/L) and fermentation temperature (37 and 55 ?C) were investigated on hydrogen production yield. Efficiency of dark hydrogen fermentation for all substrate concentrations was higher at 55 ° C compare to that at 37 °C. The hydrogen production was increased as substrate concentration increased up to 5 g-COD/L, but then it was decreased, and the optimum hydrogen yield of 38.8 mL H 2 /g-COD initial was obtained at substrate concentration and temperature of 5 g-COD/L and 55 ?C, respectively. In the third phase, the effect of initial pH (5, 6 and 7) in the production of biogas at 5 g-COD/L, and was investigated, which the highest methane yield of 213.5 mL CH 4 /g-COD initial was obtained at pH of 7. therefore, at two-stage process, the pH of fermentation and digestion were controlled to 5 and 7, respectively. Maximum amount of hydrogen and methane yield could be obtained. The hydrogen production was maximum at 5 g-COD/L (31.8 mL H 2 /g-COD initial ) compared to methane production at 3 g-COD/L (553.8 mL CH 4 /g-COD initial ) at 37 °C. The highest yield was obtained at 5 g-COD/L (35.1 mL H 2 /g-COD initial ) compared to methane production at 3 g-COD/L (95.5 mL CH 4 /g-COD initial ) at 55 °C. Maximum COD reduced of 88.1 % was afforded at substrate concentration and temperature of 3 g-COD/L and 37 °C, respectively. It was observed that increasing the temperature led to increasing biohydrogen production and decreasing in the temperature led to increasing methane production and COD removal. Keywords Bio-hydrogen, Biogas, Paper mill effluent, Dark fermentation, Anaerobic digestion