Substrates have critical effect on efficiency and cost of hydrogen production technology. Tradition evaluation index system which based on hydrogen production rate and conversion rate has limitation in comparing the economic value of different substrates utilized in hydrogen production system. This paper studies emergy of a fermentative biohydrogen production technology, comparing different biomass: wastewater and sewage sludge, the municipal solid waste and lignocellulosic biomass when they are used as substrates. Net emergy yield ratio, environmental loading ration and emergy-based sustainability index are measured. According to these indices, it shows an important role in reducing hydrogen production cost by developing cheap substrates. The results shows, the values of three indices were best when municipal wastewater was used as a substrate, it can reduce hydrogen production cost dramatically, obtain hydrogen and purify water simultaneously, benefit the environment protection.