Objective To analyze the molecular biological characteristics and antimicrobial resistance of Staphylococcus aureus (SA) isolated from hands of medical staff in Shanghai City. Methods From 2018 to 2020, medical staff of 16 different levels of hospitals in Shanghai were selected as the research objects, hands of medical staff in key departments of medical institutions were sampled, SA was isolated, identified and performed antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and whole genome sequencing, the whole genome data were assembled by BioNumerics software, the assembled genomes were conducted multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST), virulence genes, drug resistance genes and disinfec-tant resistance genes analysis. Results The isolation rate of SA from hands of medical staff in Shanghai from 2018 to 2020 was 2.9%. Methicillin-resistant SA (MRSA) accounted for 13.6% (3/22), methicillin-susceptible SA (MSSA) accounted for 86.4% (19/22). Three MRSA strains were from doctors, nurses and carers respectively, and 1 MRSA strain from doctors carried qacB gene. MSSA has different resistance to erythromycin, ciprofloxacin, tetracycline and levofloxacin; PFGE and cgMLST showed the genetic diversity of SA, PFGE bands of 2 strains of ST72 SA from the same source were highly similar, and there were only 10 differential genes, suggesting that they were clonal strains. Conclusion Hands of medical staff in medical institutions can be used as a carrier for the transmission of SA, there is transmission of SA through hands as a carrier in medical institutions.