Two-dimensional　nanomaterials　have　been　widely　used　as　fluorescent　quenching　platforms　and　nanocarriers　of　fluorophore-labelled　nucleic　acid　probes　to　design　nanoprobes　for　fluorescence　imaging　of　intracellular　microRNAs　(miRNAs).　However,　the　detection　result　was　simply　dependent　on　single　fluorescent　signal　of　fluorophore-labelled　nucleic　acid　probes.　Herein,　a　ratiometric　fluorescent　biosensor　was　proposed　by　utilizing　protonated　phenyl-doped　carbon　nitride　(PDCN)　nanosheets　not　only　as　a　fluorescent　quenching　platform　and　nanocarrier　of　ROX-ssDNA　probes　but　also　as　an　internal　fluorescent　reference.　After　specific　recognition　and　hybridization　of　target　miRNAs,　the　fluorescence　of　ROX-ssDNA　probes　was　recovered　while　the　fluorescence　of　protonated　PDCN　nanosheets　remained　unchanged.　By　using　the　fluorescence　ratio　of　ROX-ssDNA　probes　and　protonated　PDCN　nanosheets　as　the　response　signal,　this　biosensor　was　used　to　detect　DNA　analog　of　miRNA-224　over　a　linear　range　of　1–　50　nmol　L−1　with　a　limit　of　detection　0.2　nmol　L−1.　Finally,　this　ratiometric　fluorescent　biosensor　was　applied　for　fluorescence　imaging　of　miRNA-224　in　HeLa　cells　and　HepG2　cells,　which　indicated　this　biosensor　possessed　a　great　potential　for　quantitative　detection　of　miRNAs　by　fluorescence　imaging　in　living　cells.　©　2020　Elsevier　B.V.