Staphylococcus　aureus(S.　aureus)　can　cause　a　variety　of　diseases,so　it　is　essential　to　provide　the　simple　and　responsive　identification　of　bacterial　pathogens　in　clinical　treatment.　In　this　work　a　homogeneous　aptamer　electrochemiluminescence　biosensor　for　Staphylococcus　aureus　was　developed　assisted　based　on　hybridization　chain　reaction　and　magnetic　separation　assistance.　The　ds　(Probe　DNA/Aptamer)　is　modified　on　the　magnetic　beads.　In　the　presence　of　the　target　the　target　binds　to　the　Aptamer　in　ds　(Probe　DNA/Aptamer)　exposing　the　Probe　DNA　in　the　ds　DNA.　The　exposed　Probe　DNA　triggers　HCR　resulting　in　long　DNA　duplexes　which　can　be　simply　and　efficiently　enriched　for　the　luminescent　molecule　Ru(phen)32+.　To　reduce　the　possibility　of　false　positive　signals　Exo　III　was　added　to　cut　unreacted　dsDNA　from　the　magnetic　beads.　The　ECL　intensity　was　linearly　correlated　with　the　logarithm　of　S.　aureus　concentration　in　the　range　of　10　CFU/mL　−　10　×　107　CFU/mL　with　a　detection　limit　of　3　CFU/mL.　The　method　developed　in　this　work　has　good　sensitivity　and　specificity　and　can　well　evaluate　the　concentration　of　Staphylococcus　aureus　in　normal　human　serum　and　the　results　are　consistent　with　the　results　obtained　by　the　plate　counting　method.　©　2023　Elsevier　B.V.