Energy supply and environmental protection are two of the most important issues for sustainable development in today apos;s world. Due to the termination of conventional energy sources and their detrimental environmental impacts, many studies have been conducted on alternative and renewable energy sources. Among the renewable energies, hydrogen from biomass is a clean fuel with high combustion enthalpy (185 kJ / l) and as a fuel that can be replaced by fossil fuels. Various physico-chemical and biological processes are used to produce hydrogen. Among the biological methods of hydrogen production, the dark fermentation method has higher efficiency, easier control, the ability to use a variety of cheap substrates, as well as greater stability. Among the various types of reactors that used for biofuel production, biofilm reactors have been considered due to their special properties such as resistance, flexibility and high shock removal due to biofilm formation and microorganism living tissue. because of high pollution load and high oxygen demand of Industrial wastewater, it can be used as a feed in biofuels production such as biohydrogen and biogas. In the present study, the process of dark fermentation in a biofilm reactor for biohydrogen production from paper mill effluent was investigated. Initially, anaerobic sludge which was prepared from Isfahan North Wastewater Treatment Plant was used to develop microbial biofilm on the bed surfaces of the reactor. it was pretreated by heating at 90 ° C for 15 minutes. After creating the microbial biofilm, the Prepared effluent was fed to the system under control of reactor temperature and pH. Since among different operating parameters, hydraulic retention time is one of the most important factors in the performance of continuous reactors, in this study the impact of this variable in seven levels (24, 16, 12, 8, 4, 2 and 1.2 hours) was investigated on system performance and hydrogen production rate and efficiency. It was observed that by reducing the retention time till 2 hours, the rate of gas production increased to 0.68 liters of hydrogen per liter of reactor per day. The best composition of hydrogen content in the exhaust gas was 41.5% obtained at 4 h and the best hydrogen production efficiency was 0.96 mmol / g COD obtained at 8 h. As the hydraulic retention time decreased, the percentage of methane in the gas produced by the reactor was reduced So that in hydraulic retention time of 1.2 hours this amount reached 4%. It was also observed that reducing the residence time from 24 to 1.2 hours, in addition to the reducing of COD removal from 25.2 to 6% also it can be reduced the overall production of volatile fatty acids from 2.6 to 0.56 g. Due to the variation of efficiency and the ratio of volatile fatty acids produced during the 16 up 2 hour hydrolic retention time, butyric acid production pathway determined generally as the dominant pathway for the hydrogen production process in the system used. The performance of the system for producing hydrogen in the shortest retention time (1.2 h) was evaluated poor that is possibly due to the system being located near the wash-out point of microorganisms as well as feed inhibition due to its high organic loading rate. Keywords: Biohydrogen, Dark fermentation, Fixed bed biofilm reactor, Hydraulic retention time, Paper industry effluent