Introduction:　MicroRNAs　(miRNAs)　have　been　revealed　to　be　critical　genetic　regulators　in　various　physiological　processes　and　thus　quantitative　information　on　the　expression　level　of　critical　miRNAs　has　important　implications　for　the　initiation　and　development　of　human　diseases,　including　cancers.　Objectives:　We　herein　develop　three-dimensionally　(3D)　counting　of　intracellular　fluorescent　spots　for　accurately　evaluating　microRNA-21　(miRNA-21)　expression　in　individual　HeLa　cells　based　on　stimuli-activated　in　situ　growth　of　optical　DNA　flares,　grid-patterned　DNA-protein　hybrids　(GDPHs).　Methods:　Target　miRNA　is　sequence-specifically　detected　down　to　10　pM　owing　to　efficient　signal　amplification.　Within　living　cells,　GDPH　flares　are　nuclease　resistant　and　discrete　objects　with　retarded　mobility,　enabling　the　screening　of　intracellular　location　and　distribution　of　miRNAs　and　realizing　in　situ　counting　of　target　species　with　a　high　accuracy.　Results:　The　quantitative　results　of　intracellular　miRNAs　by　3D　fluorescence　counts　are　consistent　with　qPCR　gold　standard　assay,　exhibiting　the　superiority　over　2D　counts.　By　screening　the　expression　of　intracellular　miR-21　that　can　down-regulate　the　programmed　cell　death　4　(PDCD4)　protein,　the　proliferation　and　migration　of　HeLa　cells,　including　artificially-regulated　ones,　were　well　estimated,　thus　enabling　the　prediction　of　cancer　metastasis　in　murine　tumor　models.　Conclusion:　The　experiments　in　vitro,　ex　vivo　and　in　vivo　demonstrate　that　GDPH-based　3D　fluorescence　counts　at　the　single　cell　level　provide　a　valuable　molecular　tool　for　understanding　biological　function　of　miRNAs　and　especially　for　recognizing　aggressive　CTCs,　offering　a　design　blueprint　for　further　expansion　of　DNA　structural　nanotechnology　in　predicting　distant　metastasis　and　prevention　of　tumor　recurrence　after　primary　resection.　©　2022