Hydrogen is a clean energy carrier and may be made from renewable resources. Hydrogen can be produced through the butyrate/acetate or ethanol/acetate fermentation. Past researches have been carried out to identify the microbial communities from industry, food and agriculture wastewaters, which present in mixed culture and used for hydrogen production. In the present study, production of hydrogen through batch fermentation with the boiled compost of cow manure as an inoculum. Three carbohydrates including glucose, sucrose and xylose served as substrates for anaerobic fermentation to produce hydrogen. The results indicate that efficient production of hydrogen is not obtained with sole carbohydrate as substrate. The culture using mixed carbohydrates as substrates has enhanced hydrogen production under the condition at pH 4.0-4.5, Meanwhile, small amounts of acetate, ethanol and butanol were accumulated in the culture broth.