Staphylococcus aureus infection can result in a serious inflammatory condition of bone tissue, osteomyelitis with significant challenges in diagnostics. The cell surface components of the bacteria can serve as binding sites for the precise and targeted delivery of diagnostic imaging agents. We created a novel infection imaging substance using a Gallium 68 (Ga-68) labelled aptamer that specifically targets S. aureus bacteria. The targeting aptamer was selected by cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure with affinity constant at 2260 ± 634 CFU/mL. the linear detection range was between 250 and 2 x 10 4 CFU/mL. the limit of detection for s. aureus was 171 CFU/mL. The aptamer was conjugated to Ga-68 by DOTA procedure. Radioactive assays in vitro were specific for S. aureus between 10 3 and 2x10 4 CFU/mL. The encouraging outcomes indicate that this aptamer probe holds substantial promise for future in vivo experimentation, which might aid in clarifying its suitability for diagnosing S. aureus.