Detection　of　exosomes　has　aroused　great　interests　due　to　their　intrinsic　advantages　in　liquid　biopsy　for　tumor　diagnosis　and　treatment　monitoring.　Herein,　a　cascade　amplified　electrochemical　biosensor　based　on　multilegged　exosomal　walkers　was　developed　for　the　sensitive　and　selective　detection　of　exosomes.　First,　the　exosomes　were　captured　specifically　by　its　membrane　protein＇s　aptamer　and　fabricated　into　multilegged　exosomal　walkers　with　a　large　amount　of　cholesterol-labeled　DNA　strands　plunging　into　their　lipid　bilayers.　After　being　released　efficiently　by　lock　nucleic　acid　(LNA)-modified　DNA　strands　complementary　to　the　aptamers,　the　walkers　were　propelled　by　toehold　mediated　strand　displacement　reaction　(TMSDR)　and　＇walked＇　efficiently　along　DNA　＇track＇,　introducing　considerable　signal　molecules　attached　on　the　＇track＇　and　achieving　sensitive　exosomes　detection　with　a　low　limit　of　detection　(LOD)　of　29　exosomes/μL.　Owing　to　excellent　discriminative　capability　enhanced　by　LNA　and　TMSDR,　the　method　possesses　high　selectivity　toward　tumor　and　nontumor　cells　derived　exosomes,　even　for　the　serum　exosomes　from　cancer　patients　in　different　stages.　Furthermore,　the　designed　biosensor　is　reproducible.　Overall,　this　approach　provides　the　potential　to　promote　the　large-scale　development　of　liquid　biopsy　technology　with　exosomes　as　biomarkers.　©　2019　American　Chemical　Society.