The 16S rRNA genes amplification effects of the four simple total DNA extraction methods (freeze-thaw, boiling, alkaline lysis, and ROSE) were compared by extracting DNA from the Gram-negative and gram-positive bacterial strains of 14 genera. The results showed that the extraction effects of the methods from superior to inferior were in the following order: freeze-thaw, boiling, alkaline lysis and ROSE. The freeze-thaw method had advantages of cost-effective, time-and labor-saving and non-pollution. However, it was not always effective for minority gram-positive bacteria. When the DNA extraction of a large number of bacterial strains was required, the freeze-thaw method to extract total DNA of most bacteria may be first used. For the minority of DNA extraction samples which failed, it was advisable to discard the suspension from the freeze-thaw method, then use the chemical methods such as CTAB, SDS or DNA extraction kit for supplement. The freeze-thaw method to extract total DNA could not only save time and reduce cost, but also decrease the production of organic waste during DNA extrction.