An　ultrasensitive　and　specific　electrochemiluminescence　(ECL)　biosensor　has　been　designed　for　the　p53　DNA　sequence,　which　is　based　on　cascade　signal　amplification　of　nicking　endonuclease　assisted　target　recycling　and　hyperbranched　rolling　circle　amplification　(HRCA).　First　of　all,　biotin　modified　hairpin　capture　DNA　(HP)　probe　was　immobilized　on　the　surface　of　streptavidin　magnespheres　paramagnetic　particles　(PMPs).　Target　DNA　hybridized　with　the　loop　portion　of　the　HP　probe,　therefore　unfolding　HP　to　form　a　double-stranded　DNA　(dsDNA)　containing　the　specific　nicking　site　of　the　nicking　endonuclease.　Then,　the　nicking　endonuclease　recognized　the　specific　nicking　site　and　cleaved　the　HP　into　two　pieces,　liberating　target　DNA　and　the　complementary　sequence　piece　for　the　padlock　probe.　The　intact　target　DNA　would　initiate　the　next　cycle　of　hybridization　and　cleavage,　thereby　releasing　multiple　complementary　sequences　for　the　padlock　probes.　The　liberated　complementary　sequences　hybridized　with　the　padlock　probes,　subsequently　inducing　the　HRCA　reaction　and　generating　numerous　dsDNA　segments.　Herein,　Ru(phen)32+　was　embedded　into　dsDNA　and　worked　as　ECL　signal　reporter.　The　reaction　products　were　eventually　pretreated　by　dialysis　tube　with　the　cutoff　membrane　to　remove　the　residual　Ru(phen)32+　in　the　solution　for　the　following　ECL　measurements.　Using　this　cascade　amplification　strategy,　an　ultrasensitive　p53　DNA　sequence　detection　method　was　developed　with　a　wide　linear　range　from　0.05　to　100　fM　and　a　low　detection　limit　of　0.02　fM.　Moreover,　this　cascade　amplified　ECL　biosensor　had　specific　recognition　capacity　for　noncomplementary　and　single-　and　double-base　mismatched　DNA.　The　proposed　ECL　biosensor　might　have　a　great　potential　in　biomedical　research　and　clinic　analysis.　©　2016　American　Chemical　Society.