A　simple,　ultrasensitive,　and　specific　electrochemical　biosensor　was　designed　to　determine　the　given　DNA　sequence　of　Bacillus　subtilis　by　coupling　target-induced　strand　displacement　and　nicking　endonuclease　signal　amplification.　The　target　DNA　(TD,　the　DNA　sequence　from　the　hypervarient　region　of　16S　rDNA　of　Bacillus　subtilis)　could　be　detected　by　the　differential　pulse　voltammetry　(DPV)　in　a　range　from　0.1　fM　to　20　fM　with　the　detection　limit　down　to　0.08　fM　at　the　3sblank　level.　This　electrochemical　biosensor　exhibits　high　distinction　ability　to　single-base　mismatch,　double-bases　mismatch,　and　noncomplementary　DNA　sequence,　which　may　be　expected　to　detect　single-base　mismatch　and　single　nucleotide　polymorphisms　(SNPs).　Moreover,　the　applicability　of　the　designed　biosensor　for　detecting　the　given　DNA　sequence　from　Bacillus　subtilis　was　investigated.　The　result　obtained　by　electrochemical　method　is　approximately　consistent　with　that　by　a　real-time　quantitative　polymerase　chain　reaction　detecting　system　(QPCR)　with　SYBR　Green.　©　2014　American　Chemical　Society.