Abstract: In order to establish the general method for fast extracting the colony DNA from Staphylococcus aureus for PCR amplification, and compare the effects of different extraction methods on the extraction of colony DNA, the colonies of Staphylococcus aureus were treated by the boiling method, freeze-thaw method and microwave method, and the DNA was extracted. Then, through observing the effect of PCR amplification, the shortest time for obtaining DNA by the three methods and the concentration of DNA extracted after 10 s of treatment were compared. The results showed that all the three methods could successfully extract the colony DNA from Staphylococcus aureus, and were universal to the common Staphylococcus aureus. These three methods could extract the colony DNA in only 10 s, without time dependence. After 10 s of treatment, the concentration of DNA extracted by the boiling method was significantly higher than that by the freeze-thaw method and microwave method. In summary, the boiling method, freeze-thaw method and microwave method all had better results for the extraction of colony DNA from the common Staphylococcus aureus. By comparison, the boiling method was more recommended for the extraction of colony DNA.