Objective To establish and evaluate a method for rapid and sensitive Staphylococcus xylosus detection using qPCR. Methods This study selected the specific gehM gene fragment as the target for Staphylococcus xylosus, synthesized a set of specific primers and established a qPCR method for detection of Staphylococcus xylosus. Staphylococcus xylosus standard strain and other non taeget strains were chosen for specific analysis. Diluted the DNA of Staphylococcus xylosus to 10 times to determine its sensitivity. Clinical samples were tested and positive products were sequenced. The results were compared with those of bacterial culture. Results Staphylococcus xylosus had a specific amplification curve, while other non-Staphylococcus xylosus species did not show it, indicating that the primers were specific for Staphylococcus xylosus. The sensitivity was 100 fg DNA. The repeatabilities within and between groups are less than 3%. A total of 60 clinical samples were detected, of which 5 samples had a typical S curve. The PCR products were sequenced and BLAST. The similarity of the gene sequence was 99.63%, indicating that the sample was positive for the nucleic acid of Staphylococcus xylosus gehM gene, with a positive rate of 8.3%. However, the positive rate of bacterial culture was 6.7%. The positive rate of qPCR was slighterly higher than the culture. Conclusions The qPCR method established has the advantages of fast, high sensitivity and specificity, and can be used for the detection of Staphylococcus xylosus.