Objective To establish a rapid and accurate Droplet Digital PCR (ddPCR) detection method for detecting Staphylococcus aureus (SA) in experimental animals and the environment. Methods Using the heat-stable nuclease gene (nuc gene) of Staphylococcus aureus (SA) as the target gene, a pair of specific primers and probes are designed within its conserved region. Optimize the reaction conditions, test the dynamic range, and evaluate the specificity and stability of the method. Using the same template, test reactions were performed with both Droplet Digital PCR (ddPCR) and real-time quantitative PCR (qPCR) methods to assess the interchangeability between the two approaches. Finally, the method is applied to the detection of various clinical samples. Result The established SA ddPCR method has a dynamic range of 100 to 15,000 copies/μL, with a detection limit of 2.5 copies and a quantification limit of 10 copies. In testing the specificity of the method, only SA showed positive droplets, while all other pathogens showed no positive droplets. Statistical analysis of the standard deviation and relative standard deviation among three replicates revealed that within the dynamic range of digital PCR, the relative standard deviation increased as the copy number decreased, but remained below 25%, indicating that the method has good stability. A T-test analysis of the detection results from ddPCR and qPCR methods showed that there was no significant difference in the copy numbers obtained by the two methods. 10 clinical samples were tested, of which 2 were positive and the others were negative. Conclusion The established ddPCR method for detecting SA has the advantages of high sensitivity, strong specificity, good stability, and good reproducibility. This method can be applied for the detection of Staphylococcus aureus (SA) in laboratory animals.