Abstract: In order to improve the extraction method of the microbial genomic DNA from sourdough samples, the modified CTAB method, STES method and two different genomic DNA extraction kits were selected. In addition to comparing the results of the different methods by detecting the concentration and purity of the extracted microbial genomic DNA from sourdough samples, the pre-treatment method of the samples before microbial genomic DNA extraction was also optimized. The spectrophotometry and electrophoresis methods were used to determine the concentration and purity of the DNA products. Among three pre-treatment methods, including centrifugal precipitation method, washing away gluten method and drying method, centrifugal precipitation method was the best beneficial method as the pre-treatment method for microbial genomic DNA extraction from sourdough samples. In comparison with other methods, the purities of the microbial genomic DNA extracted by CTAB and STES methods were lower (D(260)/D(280)> 1.9), where the molecular weights of the extracted DNA fragment in both groups were smaller. The microbial genomic DNA may be contaminated by RNA, and there were many heterobands in the product of 16S rRNA gene when the DNA was used as the template for PCR amplification. Moreover, the ratio of D(260)/D(280) of the microbial genomic DNA extracted by the rapid DNA extraction and detection kit was less than 1.7, indicating that higher content of protein and phenolic substances in the microbial genomic DNA was present. When this kind of microbial genomic DNA was used as the template for PCR amplification, the target band of 16S rRNA gene could not obtained. Hence, this method is not suitable for the extraction of microbial gemomic DNA from sourdough either. By contrast, the integrity and the purity of the microbial genomic DNA extracted by the general genomic DNA extraction kit was the best. The concentration of microbial genome extracted from 5 g sourdough samples reached 80.10 ng/μg and had an D(260)/D(280) ratio of 1.80. Therefore, the universal genomic DNA extraction kit is the best method which is suitable for extracting microbial genomic DNA from sourdough. The results of this study can provide a method reference for the deeply further study of sourdough by molecular biological methods.