Accurate　diagnosis　of　breast　cancer　molecular　subtypes　is　crucial　for　effective　treatment　planning.　Immunohistochemistry　(IHC)　of　estrogen　receptor　(ER),　progesterone　receptor　(PR)　and　HER2　is　commonly　employed　in　clinical　diagnosis　of　breast　cancer　subtypes.　However,　IHC　has　notable　limitations,　including　the　need　of　enzymes　and　variability　in　antibody　specificity　and　sensitivity.　In　this　project,　we　developed　a　one-step　assay　technique　using　a　novel　nucleic　acid-ER/HER2　hybrid　probe-primed　hybridization　chain　reaction　(HCR)　for　detecting　ER　and　HER2　proteins　simultaneously.　The　probes　were　modified　with　specific　ligands　or　aptamers　for　ER　or　HER2　to　ensure　specificity　when　HCR　was　triggered.　This　technique　successfully　detected　ER　and　HER2　protein　expression　through　fluorescent　imaging　and　signal　quantification　in　both　cell　and　tissue　samples　in　situ　with　simple　steps.　The　detection　of　ER　and　HER2　levels　in　tissue　specimens　was　consistent　with　IHC　findings,　as　evaluated　by　ROC　analysis　(for　ER,　n　=　172,　AUC　=　0.727;　for　HER2,　n　=　174,　AUC　=　0.811).　The　HCR　method　was　a　one-step　assay　can　be　performed　quickly　without　the　need　for　enzymes,　and　the　use　of　ligands　or　aptamers-modified　probes　ensures　specificity.　Our　findings　offered　a　streamlined　and　specific　alternative　to　traditional　methods.　©　2025　Elsevier　B.V.