Abstract The combination of dark and photo fermentation was studied with cassava starch as the substrate to increase the hydrogen yield and alleviate the environmental pollution. The different raw cassava starch concentrations of 10–25 g/l give different hydrogen yields in the dark fermentation inoculated with the mixed hydrogen-producing bacteria derived from the preheated activated sludge. The maximum hydrogen yield (HY) of 240.4 ml H2/g starch is obtained at the starch concentration of 10 g/l and the maximum hydrogen production rate (HPR) of 84.4 ml H2/l/h is obtained at the starch concentration of 25 g/l. When the cassava starch, which is gelatinized by heating or hydrolyzed with α-amylase and glucoamylase, is used as the substrate to produce hydrogen, the maximum HY respectively increases to 258.5 and 276.1 ml H2/g starch, and the maximum HPR respectively increases to 172 and 262.4 ml H2/l/h. Meanwhile, the lag time (λ) for hydrogen production decreases from 11 h to 8 h and 5 h respectively, and the fermentation duration decreases from 75–110 h to 44–68 h. The metabolite byproducts in the dark fermentation, which are mainly acetate and butyrate, are reused as the substrates in the photo fermentation inoculated with the Rhodopseudomonas palustris bacteria. The maximum HY and HPR are respectively 131.9 ml H2/g starch and 16.4 ml H2/l/h in the photo fermentation, and the highest utilization ratios of acetate and butyrate are respectively 89.3% and 98.5%. The maximum HY dramatically increases from 240.4 ml H2/g starch only in the dark fermentation to 402.3 ml H2/g starch in the combined dark and photo fermentation, while the energy conversion efficiency increases from 17.5–18.6% to 26.4–27.1% if only the heat value of cassava starch is considered as the input energy. When the input light energy in the photo fermentation is also taken into account, the whole energy conversion efficiency is 4.46–6.04%.