A　highly　sensitive　homogeneous　electrochemiluminescence　(ECL)　biosensor　for　folate　receptor　(FR)　has　been　developed　based　on　hyperbranched　rolling　circle　amplification　(HRCA)　and　terminal　protection.　FR　was　combined　to　folic　acid　modified　single　stranded　DNA　(FA-ssDNA)　through　the　strong　affinity　between　FA　and　FR,　which　prevented　ssDNA　from　enzymolysis　of　exonuclease　I　(Exo　I).　The　protected　ssDNA　was　then　applied　as　a　template　of　padlock　looping　to　trigger　the　HRCA　proceeding.　The　products　of　HRCA　contains　large　amount　of　double　strand　DNA　(dsDNA),　which　provided　excellent　carries　for　Ru(phen)　3　2+　intercalation.　The　extra　free　Ru(phen)　3　2+　was　removed　by　ultrafiltration　and　the　Ru(phen)　3　2+　embedded　into　dsDNA　can　act　as　a　high-efficiency　ECL　indicator　to　quantify　the　FR　concentration.　The　enhanced　ECL　intensity　had　a　linear　relationship　with　logarithm　of　FR　concentration　in　the　range　of　4　fM　to　120　pM　with　a　correlation　coefficient　of　0.998.　Furthermore,　the　proposed　ECL　biosensor　has　been　applied　to　detect　FR　in　Hela　cells　and　showed　a　linear　relationship　with　the　logarithm　of　cell　concentration　ranging　from　100–5000　cell/mL.　Compared　to　other　FR　biosensors,　the　proposed　ECL　biosensor　presents　the　merits　of　the　simplicity,　high　sensitivity　and　specificity,　which　shows　the　great　potential　application　in　the　field　of　early　cancers　diagnosis.　©　2019　Wiley-VCH　Verlag　GmbH　&　Co.　KGaA,　Weinheim