Multiplex　DNA　methylation　and　glycosylation　are　ubiquitous　in　the　human　body　to　ensure　the　normal　function　and　stability　of　the　genome.　The　methyltransferases　and　glycosylases　rely　on　varied　enzymes　with　different　action　mechanism,　which　still　remain　challenges　for　multiple　detection.　Herein,　we　developed　a　tri-functional　dsDNA　probe　mediated　exponential　amplification　strategy　for　sensitive　detection　of　human　DNA　(cytosine-5)　methyltransferase　1　(Dnmt1)　and　uracil-DNA　glycosylase　(UDG)　activities.　The　tri-functional　dsDNA　probe　was　rationally　designed　with　M-DNA　and　U-DNA.　M-DNA　contains　the　5′-GCmGCGC-3′　site　for　Dnmt1　recognition.　U-DNA　possesses　one　uracil　as　the　substrate　of　UDG　and　a　primer　sequence　for　initiating　the　amplification　reaction.　M-DNA　was　complementary　to　partial　sequence　of　U-DNA.　In　the　presence　of　Dnmt1　and　UDG,　BssHⅡ　and　Endo　Ⅳ　were　used　to　nick　the　5′-GCGCGC-3′　and　AP　sites　respectively,　resulting　in　the　release　of　single-stranded　DNA　sequence　(primer　sequence),　respectively.　After　magnetic　separation,　the　released　primer　sequence　hybridizes　with　padlock　DNA　(P-DNA),　initiating　exponential　rolling　circle　amplification　to　produce　numerous　G-quadruplexes　for　recordable　signals.　The　strategy　exhibited　the　limit　of　detection　as　low　as　0.009　U　mL−1　and　0.003　U　mL−1　for　Dnmt1　and　UDG,　respectively.　Meanwhile,　this　strategy　was　successfully　applied　to　detect　Dnmt1　and　UDG　activities　in　living　cell　samples　at　single-cell　level　and　assay　the　inhibitors　of　Dnmt1　and　UDG.　Therefore,　the　strategy　provided　a　potential　method　to　detect　Dnmt1　and　UDG　activities　in　biological　samples　for　early　clinic　diagnosis　and　therapeutics.　©　2019　Elsevier　B.V.