Single　nucleotide　polymorphisms　(SNPs)　present　significant　challenges　in　microbial　detection　and　treatment,　further　raising　the　demands　on　sequencing　technologies.　In　response　to　these　challenges,　we　have　developed　a　novel　barcode-based　approach　for　highly　sensitive　single　nucleotide　recognition.　This　method　leverages　a　dual-head　folded　complementary　template　probe　in　conjunction　with　DNA　ligase　to　specifically　identify　the　target　base.　Upon　recognition,　the　system　triggers　rolling　circle　amplification　(RCA)　followed　by　the　self-assembly　of　CdSe　quantum　dots　onto　polystyrene　microspheres,　enabling　a　single-particle　fluorescence　readout.　This　approach　allows　for　precise　base　identification　at　individual　loci,　which　are　then　analyzed　using　a　bio-barcode　array　to　screen　for　base　changes　across　multiple　sites.　This　method　was　applied　to　sequence　a　drug-resistant　mutation　site　in　Helicobacter　pylori　(H.　pylori),　demonstrating　excellent　accuracy　and　stability.　Offering　high　precision,　high　sensitivity,　and　single　nucleotide　resolution,　this　approach　shows　great　promise　as　a　next-generation　sequencing　method.　©　2024　by　the　authors.