An　ultrasensitive　electrochemiluminescence　(ECL)　aptamer　sensor　for　protein　(thrombin　as　an　example)　detection　based　on　hyperbranched　rolling　circle　amplification　(HRCA)　had　been　developed.　A　complementary　single-strand　DNA　(CDNA)　of　the　thrombin　aptamer　had　been　modified　on　the　gold　electrode　firstly,　and　then　hybridized　with　thrombin　aptamer　to　make　the　aptamer　immobilized　on　the　electrode　surface,　in　the　presence　of　thrombin,　aptamer-thrombin　bioaffinity　complexes　formed　and　made　thrombin　aptamer　leave　the　electrode　surface.　Thus,　the　linear　padlock　probe　hybridized　with　the　free　CDNA　on　the　electrode　surface　and　circularized　by　Escherichia　coli　DNA　ligase.　Subsequently,　the　linear　padlock　probe　was　served　as　a　template　for　the　initiation　of　HRCA　reaction,　and　a　lot　of　dsDNA　modified　on　the　electrode　surface.　Then　Ru(phen)(3)(2+)　(acted　as　the　ECL　indicator)　intercalates　specifically　into　double-stranded　DNA　(dsDNA)　grooves　to　generate　ECL　signal.　The　ECL　intensity　of　the　system　has　a　linear　relationship　with　thrombin　concentration　in　the　range　of　3.0-300　aM　with　a　detection　limit　of　1.2　aM　(S/N=3).　The　proposed　method　combines　the　high　sensitivity　of　ECL,　exponential　amplification　of　HRCA　for　signal　enhancement　and　high　selectivity　of　aptamer.　(C)　2014　Elsevier　B.V.　All　rights　reserved.