In this study, a sonication-based DNA extraction method was developed, in which the whole process can be finished within 10 min. This method is almost zero cost and time-saving, which is useful for high throughput screening, especially in the screening of mutants generated in random mutagenesis. This method is effective in genomic DNA extraction for PCR amplification in several Gram-positive bacteria, including Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Listeria monocytogenes.