Breast　cancer　is　the　most　frequently　diagnosed　cancer　among　women,　and　the　circulating　tumor　cell　(CTC)-meditated　distant　metastasis　is　the　leading　cause　of　death.　Thus,　the　detection　of　CTCs　is　of　great　importance　for　the　early　diagnosis　of　breast　cancer　and　the　prevention　of　metastasis.　In　this　study,　using　human　breast　carcinoma　BT474　cells　as　the　model　CTCs,　a　powerful　assay　platform　is　demonstrated　by　fluorescence　spectrometry　for　the　highly　sensitive　CTC　detection　by　combining　the　dual-recognizing　elements　receptor-binding　antibody　and　aptamer-mediated　separation　with　double　rolling　circle　amplification　reactions　(d-RCA,　including　RCA1　and　RCA2).　The　aptamer-inserted　RCA1　product　(RCA1-p)　exhibits　the　considerably　improved　affinity　towards　target　cells　originating　from　the　multivalent　binding　effect.　The　immunomagnetic　separation　removes　nontarget　cells　coexisting　in　complex　biological　milieu,　while　the　centrifugal　separation　of　cells/DNAs　mixture　eliminates　the　excess　probes,　thereby　circumventing　the　unwanted　interferences.　The　fluorescence　spectrometric　results　show　that　a　34-fold　enhanced　fluorescence　signal　is　achieved　upon　BT474　cells,　and　the　target　cells　can　be　quantitatively　detected　down　to　9　cells/200　mu　L　with　the　linear　range　of　five　orders　of　magnitude,　indicating　a　significantly　enhanced　detection　performance.　Even　if　BT474　cells　are　spiked　in　the　fresh　whole　blood,　no　obvious　fluctuation　in　the　fluorescence　signal　is　detected,　demonstrating　that　the　newly　developed　d-RCA　assay　system　is　suitable　for　screening　CTCs　in　complex　environments　and　is　expected　to　be　a　promising　tool　for　estimating　distant　metastasis　and　predicting　the　recurrence　of　tumors.