This study investigates the characteristics of an anaerobic fermentation for hydrogen yield, using sewage sludge and simulated food residues as substrates inoculate with cow manure. The moisture content was controlled at less than 80% to promote the concentration of substrates and minimize the generation of fermentation liquid, thus eliminate the subsequent treatment problem. The effects of fermentation temperature on hydrogen yield were characterized by batch fermentation between 50°C and 75°C, separated by 5°C increments. The results indicate that the 60 oC batch showed specific hydrogen yield and specific hydrogen conversion rate were relatively high, 39 mL-H2/g-VS and 4.00 g-H2/g-H of VS%, respectively. However, the average hydrogen generation rate, measured around the exponential growth phase of the batch, occurred at 65 oC batch. Furthermore, in fermentation experiments with mixed substrates at 60 oC and 65 oC series, both have an optimum mix ratio by weight, the ratio of sludge, simulated food residues and cow manure were found to be 2 : 1 : 1. Herein 60 oC series has higher cumulative volume than 65 oC series in this study. Pretreatment of the fermentation substrates by acid, alkali, heating, and agitation may affect the hydrogen fermentation characteristics. The results indicate except acid treatment poor for it, the others can enhance the hydrogen yield.