Objective To establish a rapid and accurate droplet digital PCR (ddPCR) detection method for detecting Staphylococcus aureus (SA) in laboratory animals and the environment. Methods Using the heat-stable nuclease gene (nuc) of 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 ddPCR and real-time quantitative PCR (qPCR) method to assess the interchangeability between the two approaches. Finally, the method is applied to the detection of various clinical samples. Results The kinetic range of the established SA ddPCR method is 100 ~15 000 copies/ μL, with a detection limit of 2. 5 copies and a quantification limit of 10 copies; The specificity of this method was tested, and only SA showed positive droplets, while no positive droplets were found for other pathogens;After measuring three parallel samples, the standard deviation and relative standard deviation were calculated. It was found that within the dynamic detection interval of ddPCR, as the target copy number gradually decreased, the relative standard deviation showed an upward trend, but remained below 25%. This result indicates that the detection method has good stability. Conclusions 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 SA in laboratory animals.