The effects of Fe and Fe2+ concentration in the external environment on hydrogen production capacity of ethanol-type fermentative microorganism flora were investigated. The optimum range of Fe and Fe2+ concentration enhanced hydrogen production were obtained and the characteristic of fermentation types at different concentration of Fe and Fe2+ were discussed. It was demonstrated that both Fe and Fe2+ were able to enhance the hydrogen production capacity of microorganism flora. The maximum hydrogen production yield of 143.7mL x g(-1) glucose and the maximum specific hydrogen production rate 21.2mL x (h x g)(-1) were obtained at Fe2+ concentration of 200mg x L(-1) and 50mg x L(-1). The maximum hydrogen production yield of 156.1mL x g(-1) glucose and the maximum specific hydrogen production rate 23.5mL x (h x g)(-1) were obtained at Fe concentration of 1000mg x L(-1) and 500mg x L(-1). At the same concentration, the enhancement of Fe is better than Fe2+ when the concentration is higher than 50 mg x L(-1). A sequential extraction technique was used to represent arbitrary divisions between different metal forms of Fe2+ in the mixed culture.