Abstract: It is required to efficiently extract large quantity and high quality of fungal DNA for the soil fungal molecular ecological study. However, the yield and quality of DNA are often insufficient or even no any product can be produced by the commercial DNA extraction kit. To solve these problems, the cell lysis and subsequent DNA extraction parameters were optimized in this study, and then an efficient method of extracting soil fungal DNA was established. The results showed this method was very sensitive to entomopathogenic fungi Verticillium lecanii. DNA could be extracted from the pure culture samples which contained more than 10 spores, and the artificial soil samples which contained more than 100 spores per gram soil. However commercial DNA extraction kits are unable to extract DNA from the pure culture samples which contained 10⁷ spores. The DNA product from our method did not show any obvious inhibition in the subsequent PCR amplification, and was proved to be high-pure by the OD_260/280 value. DNA could be also extracted from - two natural soil samples,but the target sequence fragment specific for V. lecanii was only amplified from one natural soil sample applied with V. lecanii. In conclusion, this soil DNA extraction method is sensitive to entomopathogenic fungi and DNA products were high-pure, so it was suitable for the DNA preparation in soil entomopathogenic fungal molecular ecological research.