The　detection　of　specific　DNA　sequences　and　the　identification　of　single　nucleotide　polymorphisms　are　important　for　disease　diagnosis.　Herein,　by　combining　the　high　specificity　of　the　base-stacking　effect　with　the　high　reproducibility　of　bovine　serum　albumin　(BSA)　modified　electrodes　and　the　high　loading　performance　of　DNA　nanoclews　(DNA　NCs),　a　novel　sandwich-type　electrochemiluminescence　(ECL)　biosensor　is　reported　for　the　highly　specific　detection　of　HPV16　(chosen　as　the　model　target).　The　capture　probes　are　loaded　by　BSA　carrier　platforms　modified　on　the　gold　electrode　surface　to　improve　reproducibility.　DNA　NCs　loaded　with　a　large　amount　of　Ru(phen)3　2+　worked　as　signal　probes.　The　template　probe　is　composed　of　the　complementary　strand　of　the　target　and　two　free　nucleic　acid　anchors　at　the　head　and　tail.　In　the　presence　of　the　target　DNA,　the　template　probes　can　form　stacked　base　pairs　with　target,　generating　high　base-stacking　energy.　This　results　in　the　shorter　free　anchors　of　template　probes　being　able　to　bind　to　the　capture　and　signal　probes.　This　eventually　forms　a　sandwich　structure　that　allows　Ru(phen)3　2+　to　be　near　the　electrode　surface,　producing　an　ECL　signal.　There　is　a　linear　relationship　between　the　signal　and　the　target　concentration　range　from　10　fM　to　100　pM,　with　a　detection　limit　of　5.03　fM　(S/N=3).　Moreover,　the　base-stacking　effect　has　single　base　recognition　ability　for　base　pairs,　effectively　avoiding　false　positive　signals.　The　results　of　this　strategy　for　clinical　samples　are　consistent　with　classical　methods.　©　2023　Elsevier　B.V.