This article discusses the method of producing hydrogen from water hyacinth. Water hyacinth was pretreated with microwave heating and alkali to enhance the enzymatic hydrolysis and hydrogen production in a two-step process of dark- and photo- fermentation. Water hyacinth with various concentrations of 10–40 g/l was pretreated with four methods: (1) steam heating; (2) steam heating and microwave heating/alkali pretreatment; (3) steam heating and enzymatic hydrolysis; (4) steam heating, microwave heating/alkali pretreatment and enzymatic hydrolysis. Water hyacinth (20 g/l) pretreated with method 4 gave the maximum reducing sugar yield of 30.57 g/100 g TVS, which was 45.6% of the theoretical reducing sugar yield (67.0 g/100 g TVS). The pretreated water hyacinth was used to produce hydrogen by mixed H₂-producing bacteria in dark fermentation. The maximum hydrogen yield of 76.7 ml H₂/g TVS was obtained at 20 g/l of water hyacinth. The residual solutions from dark fermentation (mainly acetate and butyrate) were used to further produce hydrogen by immobilized Rhodopseudomonas palustris in photo fermentation. The maximum hydrogen yield of 522.6 ml H₂/g TVS was obtained at 10 g/l of water hyacinth. Through a combined process of dark- and photo- fermentation, the maximum hydrogen yield from water hyacinth was dramatically enhanced from 76.7 to 596.1 ml H₂/g TVS, which was 59.6% of the theoretical hydrogen yield.