Abstract: The aim of this study was to establish a fingerprint of volatile metabolites from S. Aureus according to the volatile characteristics of Staphylococcus aureus. In the 7.5% NaCl broth medium, volatile metabolites of S. aureus were detected and analyzed using headspace solid phase microextraction/gas chromatography-mass spectrometry (HS-SPME-GC/MS) and automated mass spectral deconvolution and identification system (AMDIS), combined with similarity evaluation system for chromatographic fingerprints of TCM. Research established a mass spectrometry database consisting of 17 target volatile metabolites of S. aureus was established, and based on this database, the similarities of 10 sets of chromatograms were all above 0.90, which met the technical requirements of fingerprints. Based on this, a fingerprint of volatile metabolites of S. aureus was established. The match and reverse march of the compounds identified by the HS-SPME-GC/MS-AMDIS in NIST were all above 800, and the minimum match factor were all above 80, hence the results are accurate, reliable and easy to operate. The established fingerprint of volatile metabolites of S. aureus could be used as a reference for the quality monitoring and analysis of S. aureus.