This　work　designs　a　novel　programmable　hairpin　probe　(PHP)　for　the　immobilization-free　electrochemical　detection　of　nucleic　acid　by　coupling　polymerase/nicking-induced　isothermal　signal　amplification　strategy　with　a　biphasic　reaction　mode　for　the　first　time.　The　designed　PHP　(including　a　target-recognition　region,　a　template　sequence　for　enzymatic　reaction　and　an　inactivated　anti-streptavidin　aptamer)　could　program　multiple　isothermal　reactions　in　the　solution　phase　accompanying　in　situ　amplified　detectable　signal　at　the　electrode　surface　by　the　labeled　ferrocene　tag　on　the　PHP.　Upon　addition　of　target　analyte　into　the　detection　solution,　target　DNA　initially　hybridized　with　the　recognition　region　on　the　PHP.　Replication-induced　strand-displacement　generated　an　activated　anti-streptavidin　aptamer　with　the　assistance　of　polymerase.　Then,　the　polymerase/nicking　enzymes　could　cleave　and　polymerize　repeatedly　the　replication　product,　thus　resulting　in　the　formation　of　numerous　template-complementary　DNA　initiator　strands.　The　released　initiator　strands　could　retrigger　the　programmable　hairpin　probe　to　produce　a　large　number　of　activated　anti-streptavidin　aptamers,　which　could　be　captured　by　the　immobilized　streptavidin　on　the　electrode,　thus　activating　the　electrical　contact　between　the　labeled　ferrocene　and　the　electrode.　Going　after　the　aptamers,　the　carried　ferrocene　could　produce　the　in　situ　amplified　electronic　signal　within　the　applied　potentials.　Under　optimal　conditions,　the　electrochemical　signal　increased　with　the　increasing　target　DNA　concentration　in　the　dynamic　range　from　5　fM　to　10　pM　with　a　detection　limit　(LOD)　of　2.56　fM　at　the　3sblank　criterion.　Importantly,　the　methodology　with　high　specificity　was　also　validated　and　evaluated　by　assaying　6　target　DNA-spiked　human　serum　and　calf　thymus　DNA　samples,　and　the　recoveries　were　95-110%.　Further　work　for　the　programmable　hairpin　probe　could　be　even　utilized　in　a　real　world　sample　to　detect　miRNA-21　at　femtomol　level.　©　2014　American　Chemical　Society.