Abstract: The improved Hungate rolling tube technique, the techniques about plate of anaerobic operation and the anaerobic method using plate culture flask are used to isolate H2-producing bacteria. And these work are very tedious. If the material, used for test, was pretreated for the purpose of selective culture, the work load will be reduced in the next steps.The way of heat treatment, the acid treatment and the techniques about plate of anaerobic operation and plate of culture bottle were adopted in this study to isolate one strain of H2-producing bacteria. And this germ has high ability of H2-producing during the 45℃ to 50℃, rod-shape, and no flagellum. It was eligible to treat the sludge for 10 minutes under the condition of 85℃ to isolate H2-producing bacteria.