Bacteria originating from the hospital environment could lead to infection on humans and have a high risk of nosocomial infection. This study aims to identify and detection of DNA plasmid in Staphylococcus aureus (S. aureus), which comes from the hand swabs and swab clinical nurses in hospitals of Jayapura, Papuan Province of Indonesia. In this study were found 96 isolates of S. aureus. Identification of S. aureus conducted through fermentation test of mannitol salt agar (MSA), gram staining, coagulase test and catalase test. Here, we showed that all isolates of S. aureus were able to ferment MSA, coagulase and catalase positive test. Results of resistance on 96 isolates of Staphylococcus aureus on sensitivity test to antibiotics oxacilin 89.6%, vankomicin 47.9%, ampicillin 79.2%, tetraciklin 46.9% and gentamicin and norfloxacin are 0%. Results of plasmid isolation and electrophoresis test, in get 3 bands with the size of 9.5 kb DNA using Lambda Hind III DNA as marker. From the results of this study concluded that the high levels of Staphylococcus aureus bacteria resistant to antibiotics and allegedly mediated by plasmid DNA at Jayapura hospital, Papua-Indonesia