Two-dimensional　(2D)　nanomaterials　have　been　extensively　used　as　quenching　platforms　of　fluorescently　labeled　oligonucleotides　for　the　design　of　biosensors.　However,　not　all　fluorophore-labeled　single-stranded　DNA　(ssDNA)　is　necessarily　quenched　after　they　are　adsorbed　on　the　surface　of　2D　nanomaterials.　Herein,　we　discovered　that　the　fluorescence　of　tetramethylrhodamine　(TMR)-labeled　ssDNA　was　not　quenched　but　enhanced　by　polymeric　carbon　nitride　nanosheets　(PCN　NSs).　Effects　of　the　fluorophore,　oligonucleotide　sequence,　and　the　number　of　proximal　G　bases　on　the　fluorescence　of　fluorophore-labeled　ssDNA　were　investigated　in　the　presence　of　PCN　NSs.　The　fluorescence　enhancement　phenomena　were　not　observable　for　fluorescein-　and　X-rhodamine-labeled　ssDNA　and　other　2D　nanomaterials　such　as　graphene　oxide　and　MoS2NSs.　In　the　presence　of　PCN　NSs,　steric　hindrance　effect　shields　the　photoinduced　electron　transfer　quenching　effect　between　TMR　and　proximal　G　bases,　which　may　account　for　the　fluorescence　enhancement　of　TMR-labeled　ssDNA　adsorbed　on　PCN　NSs.　Benefiting　from　the　fluorescence　enhancement　effect　and　the　affinity　change　of　PCN　NSs　to　ssDNA　probes　upon　hybridization　with　complementary　ssDNA,　a　versatile　ratiometric　biosensing　platform　as　a　proof　concept　was　demonstrated　for　the　high　sensitive　fluorescence　detection　of　ssDNA　and　microRNA.　©　2023　American　Chemical　Society.　All　rights　reserved.