Our　preliminary　investigation　has　identified　the　potential　of　serum　fucosylated　extracellular　vesicles　(EVs)　miR-4732-5p　in　the　early　diagnosis　of　lung　adenocarcinoma　(LUAD)　by　a　fucose-captured　strategy　utilizing　lentil　lectin　(LCA)-magnetic　beads　and　subsequent　screening　of　high　throughput　sequencing　and　validation　of　real-time　quantitative　polymerase　chain　reaction (RT-qPCR).　Considering　the　relatively　complicated　procedure,　expensive　equipment,　and　stringent　laboratory　condition,　we　have　constructed　an　electrochemical　biosensor　assay　for　the　detection　of　miR-4732-5p.　miR-4732-5p　is　extremely　low　in　serum,　down　to　the　fM　level,　so　it　needs　to　be　detected　by　highly　sensitive　electrochemical　methods　based　on　the　Mg2+-dependent　DNAzyme　splitting　nucleic　acid　lock　(NAL)　cycle　and　hybridization　chain　reaction　(HCR)　signal　amplification.　In　this　study,　signal　amplification　is　achieved　through　the　dual　amplification　reactions　using　NAL　cycle　in　combination　with　HCR.　In　addition,　hybridized　DNA　strands　bind　to　a　large　number　of　methylene　blue　(MB)　molecules　to　enhance　signaling.　Based　on　the　above　strategy,　we　further　enhance　our　signal　amplification　strategies　to　improve　detection　sensitivity　and　accuracy.　The　implementation　of　this　assay　proceeded　as　follows:　initially,　miR-4732-5p　was　combined　with　NAL,　and　then　Mg2+-dependent　DNAzyme　splitted　NAL　to　release　auxiliary　DNA　(S1)　strands,　which　were　subsequently　captured　by　the　immobilized　capture　probe　DNA　(C1)　strands　on　the　electrode　surface.　Following　this,　abundant　quantities　of　DNA1　(H1)　and　DNA2　(H2)　tandems　were　generated　by　HCR,　and　S1　strands　then　hybridized　with　the　H1　and　H2　tandems　through　base　complementary　pairing.　Finally,　MB　was　bonded　to　the　H1　and　H2　tandems　through　π–π　stacking　interaction,　leading　to　the　generation　of　a　signal　current　upon　the　detection　of　a　potential　capable　of　inducing　a　redox　change　of　MB　by　the　electrode.　Furthermore,　we　evaluated　the　performance　of　our　developed　electrochemical　biosensor　assay.　The　results　demonstrated　that　our　assay　is　a　reliable　approach,　characterized　by　its　high　sensitivity　(with　a　detection　limit　of　2.6　×　10−17 M),　excellent　specificity,　good　accuracy,　reproducibility,　and　stability.　Additionally,　it　is　cost-effective,　requires　simple　operation,　and　is　portable,　making　it　suitable　for　the　detection　of　serum　fucosylated　extracellular　vesicles　miR-4732-5p.　Ultimately,　this　development　has　the　potential　to　enhance　the　diagnostic　efficiency　for　patients　with　early-stage　LUAD.　©　The　Author(s)　2024.