Abstract: How to get the total DNA of rumen microbes was the key factor on molecular research. The molecular size of the DNA of rumen microbes was very great, maximum size is 15 to 20kb. Therefore, an optimized method to get total DNA should be chosen for the future research. In this paper, some physical disruption methods were chosen, such as glass bead mill homogenization method, freeze-thaw treatment, ultrasonic lysis treatment, liquid N2 mill treatment etc, and some chemical method, such as sodium dodecyl sulfate solution, Cetyltrimethyl Ammonium Bromide solution and lysozyme pretreatment (lysozyme, proteinase K). And they were compared to choose the most optimized one to fit next PCR and other research. The results showed the total DNA quality of rumen microbes with the glass bead mill homogenization plus CTAB extraction solution method and the freeze-thaw treatment plus SDS extraction solution method were better than that with other methods, moreover, the sample of the total DNA can be used in the molecular experiment through electrophoresis and PCR detection.