A　ratiometric　electrochemical　aptasensor　based　on　gold　nanoparticles　(AuNPs)　functionalization　and　hybridization　chain　reaction　(HCR)　assisted　signal　amplification　has　been　for　the first time　designed　for　the　detection　of　streptomycin　(STR).　The　double-stranded　DNA　(dsDNA)　formed　by　the　hybridization　of　ferrocene　(Fc)-labeled　STR　aptamer　(Apt)　and　capture　probe　(CP)　is　first　immobilized　on　the　gold　electrode　(GE)　surface　via　Au–S　reaction.　The　specific　binding　of　the　target　and　Apt　results　in　numerous　Fc　detachment　from　the　sensing　interface.　Then,　the　remaining　single-stranded　CP　is　combined　with　AuNPs　modified　with　initiator　DNA　(iDNA)　by　auxiliary　DNA　(aDNA).　Among　them,　the　iDNA　triggers　HCR　between　two　hairpin　probes　(H1/H2),　thus　capturing　a　large　number　of　methylene　blue　(MB)　electrochemical　probe,　which　generates　a　strong　electrochemical　signal　of　MB　and　a　weak　electrochemical　signal　of　Fc.　Signals　are　collected　by　square　wave　voltammetry　(the　potential　window　ranging　from　-0.5 V　to　0.6 V,　vs. Ag/AgCl),　and　the　oxidation　peak　currents　at　-0.200 V　(MB)　and　0.416 V　(Fc)　are　recorded.　The　use　of　the　ratiometric　method　has　effectively　improved　the　accuracy　and　reliability　of　the　analysis.　The　successful　application　of　AuNPs　and　HCR　greatly　improves　the　sensitivity　of　the　sensor,　and　the　detection　limit　is　as　low　as　0.08 pM.　It　can　sensitively　determine　STR　in　the　range　0.1 pM　to　10 nM.　In　addition,　the　designed　aptasensor　has　been　successfully　applied　to　the　detection　of　STR　in　milk　and　honey　samples.　Graphical　Abstract:　[Figure　not　available:　see　fulltext.].　©　2023,　The　Author(s),　under　exclusive　licence　to　Springer-Verlag　GmbH　Austria,　part　of　Springer　Nature.