Recently, the use of hydrogen as a clean energy source in industry has been increased. Compared to conventional hydrogen production methods, biological hydrogen production methods are characterized by being less energy intensive, non- polluting and low-value substrates (waste substrates) can be used for the bio-H2 production. Among different biological methods, dark fermentation has gained more attention because the process can be conducted in absence of light and a variety of organic substrates can be utilized. The main drawback of the dark fermentation process is the low H2 yield; this occurs because of the biomass washout in case of using CSTR bioreactor and low biodegradation substrates such as food waste, agricultural residues and/or industrial wastewaters that contain high complex pollutants e.g. textile wastewater, pesticides wastewater, etc. In order obtain high H2 yields, the efficiency of the fermentation process must be enhanced by maintaining high biomass concentration in the bioreactor and improving the biodegradation of the used organic wastes. The cell density increased in the bioreactor by formation of granular sludge using sucrose wastewater. Different sucrose concentrations of 10–30 g/L were studied, with 5 g/L increment. Although the HPR increased with increasing sucrose concertation in the feed, optimum hydrogen yield of 361.1 NmL-H2/g-sucrose was obtained at 10 g/L and the H2 yields decreased at higher sucrose concentrations. The hydrogen yield from sucrose wastewater was enhanced by using two-stage process such as dark/dark and/or dark/photo-fermentation systems. In case of dark/dark combined system, the hydrogen yield increased from 2.14 (one-stage) to 4.20 mol-H2/mol-sucrose (two-stage). Likewise, the hydrogen yield increased from 2.64 mol-H2/mol-sucrose in one-stage (dark fermentation) to 4.84 mol-H2/mol- sucrose when dark/photo-fermentation system was used. In case of agricultural residues, several pre-treatment methods including heat, ultrasonication, alkaline, acid, hydrogen peroxide were applied alone or in combination to enhance the biohydrogen production from potatoes and bean wastes in batch and continuous experiments. The H2 yields were higher using pre-treated samples than the corresponding yields achieved using the raw substrates e.g. potatoes and bean wastes. For both substrates, the best H2 yields were observed using H2O2 pre-treated wastes, while low H2 yields were measured in case of heat and ultrasonic pre-treatments. The biohydrogen production from starch-containing textile wastewater (TWW) was enhanced by application of photocatalytic degradation and Fenton oxidation pre- treatments. The H2 yield increased from 157.9 NmL-H2/g-VS using the raw TWW to 169.4–284.0 and 186.9–304.1 NmL-H2/g-VS using photocatalytic degradation and Fenton oxidation pre-treatments, respectively.