16S　ribosomal-RNA　(16S　rRNA)　is　often　used　as　an　ultrasensitive　marker　for　Chlamydia　trachomatis　(CT)　detection　because　of　its　species　specificity　and　high　copy　number　in　CT.　Robust　methods　for　16S　rRNA　detection　must　be　developed　to　realize　the　early　diagnosis　of　CT　infections.　In　this　work,　a　highly　reproducible　and　sensitive　electrochemical　biosensor　based　on　duplex-specific　nuclease　(DSN)-assisted　target-responsive　DNA　hydrogels　and　bovine　serum　albumin　(BSA)　carrier　platform　for　CT　detection　was　developed.　Target　rRNA　can　trigger　the　DNA　hydrogel　response,　which　causes　it　to　be　repeatedly　cleaved　by　DSN,　ultimately　leading　to　the　release　of　a　large　amount　of　horseradish　peroxidase-labelled　streptavidin　(SA-HRP)　embedded　in　the　hydrogel　beforehand.　The　released　SA-HRP　was　stably　captured　by　the　capture　probes　that　were　orderly　loaded　at　the　gold　electrode　with　the　help　of　a　BSA　layer.　Then,　SA-HRP　catalyzed　the　redox　reaction　of　3,3′,5,5′-tetramethylbenzidine　and　H2O2,　producing　a　current　signal　that　can　be　detected.　The　current　signal　was　proportional　to　the　concentration　of　CT　16S　rRNA　from　10　fM　to　25　pM　with　a　detection　limit　of　5.8　fM　(S/N　=　3).　The　signal　conversion　function　of　the　DNA　hydrogel　avoids　the　instability　of　nonhomogeneous　nucleic　acid　hybridization　on　the　gold　electrode　surface,　and　combined　with　optimization　by　BSA　for　capture　probe　modification,　this　electrochemical　biosensor　is　highly　reproducible　with　a　relative　standard　deviation　of　4.3%　for　the　detection　of　10　samples　of　the　same　concentration.　The　proposed　strategy　provides　a　highly　reproducible　and　sensitive　detection　method　for　the　extensive　screening　of　CT.　©　2022　Elsevier　B.V.