Traditional　electrochemical　DNA　biosensors　need　DNA　immobilization　on　the　electrode　surface,　which　is　tedious　and　time-consuming.　In　this　study,　a　simple　but　ultraselective　electrochemical　DNA　biosensor　had　been　designed　to　determine　target　DNA　species　related　to　oral　cancer　overexpressed　1　in　saliva,　which　combines　the　signal　amplification　of　nicking　endonuclease　assisted　target　recycling　with　the　immobilization-free　electrochemical　method.　The　complementary　substrate　strand　of　target　DNA　species　contains　a　simple　asymmetric　sequence　had　been　modified　with　a　methylene　blue　at　the　3′　terminal　first,　which　cannot　diffuse　easily　to　the　negative　charged　ITO　electrode　surface　due　to　the　abundant　negative　charges.　The　presence　of　the　target　DNA　would　trigger　the　formation　of　double-stranded　DNA　(dsDNA).　Then　the　nicking　endonuclease　can　recognize　the　simple　asymmetric　sequence　in　the　dsDNA　and　cleave　the　substrate　strand　of　ds-DNA　into　two　pieces,　a　long　ssDNA　and　a　2-base　ssDNA　linked　with　methylene　blue.　The　short　one　can　diffuse　easily　to　the　negative　charged　ITO　electrode　surface　and　results　in　the　enhanced　electrochemical　response　detected.　At　the　same　time,　the　target　DNA　can　dissociate　from　the　dsDNA　and　trigger　the　next　round　of　hybridization,　cleavage,　and　releasing,　which　results　in　the　signal　amplification.　This　homogeneous　DNA　biosensor　can　detect　as　low　as　0.35　pM　(S/N　=　3)　target　DNA.　Compared　with　the　traditional　heterogeneous　electrochemical　DNA　biosensors,　which　are　tedious　and　time-consuming　due　to　the　complex　DNA　immobilization　process,　the　assay　not　only　owns　the　merits　of　simple　and　high　efficiency　since　performed　in　a　homogeneous　solution　but　also　exhibits　a　high　distinction　ability　to　single-base　mismatch,　double-bases　mismatch,　and　noncomplementary　DNA　sequence.　©　2015　American　Chemical　Society.