Exosomes　secreted　by　tumor　cells　play　a　crucial　role　in　tumor　progression　and　immune　regulation,　making　their　quantification　essential　for　early　cancer　detection　and　prevention.　In　this　paper,　we　design　a　DNA　walker　based　on　a　tetramolecular　G-quadruplex　with　a　position-controllable　swing　arm.　The　sensitive　detection　of　exosomes　was　achieved　through　the　G-quadruplex　acting　as　track　strands,　and　their　top-labeled　ferrocene　(Fc)　act　as　electrochemical　indicators,　which　were　oxidation　catalyzed　by　[Fe(CN)6]4−.　The　specific　identification　was　attributed　to　the　aptamer　that　can　capture　the　CD63　protein　on　exosomes.　The　power　of　the　swing　arm　was　caused　by　the　nucleic　acid　endonuclease　Nt.　BbvCI,　which　leads　to　the　release　of　the　Fc-labeled　DNA　fragments,　and　thus　the　electrochemical　signal　of　Fc　at　the　electrode　decreases　as　the　DNA　walker　walks.　The　electrochemical　biosensor　has　a　wide　linear　range　(40–2.5　×　106　particles/μL)　and　a　low　detection　limit　(1.88　particles/μL),　and　also　exhibits　excellent　reproducibility　and　stability.　This　strategy　provides　new　ideas　for　the　development　of　novel　DNA　biosensors　and　biological　applications　of　G-quadruplex.　©　2025　Elsevier　B.V.