DNA　glycosylase　is　an　indispensable　DNA　damage　repair　enzyme　which　can　recognize　and　excise　the　damaged　bases　in　the　DNA　base　excision-repair　pathway.　The　dysregulation　of　DNA　glycosylase　activity　will　give　rise　to　the　dysfunction　of　base　excision-repair　and　lead　to　abnormalities　and　diseases.　The　simultaneous　detection　of　multiple　DNA　glycosylases　can　help　to　fully　understand　the　normal　physiological　functions　of　cells,　and　determine　whether　the　cells　are　abnormal　in　pre-disease.　Regrettably,　the　synchronous　detection　of　functionally　similar　DNA　glycosylases　is　a　great　challenge.　Herein,　we　developed　a　multifunctional　dsDNA　probe　mediated　exponential　rolling　circle　amplification　(E-RCA)　method　for　the　simultaneously　sensitive　detection　of　human　alkyladenine　DNA　glycosylase　(hAAG)　and　uracil-DNA　glycosylase　(UDG).　The　multifunctional　dsDNA　probe　contains　the　hypoxanthine　sites　and　the　uracil　sites　which　can　be　recognized　by　hAAG　and　UDG　respectively　to　generate　apyrimidinic　(AP)　sites　in　the　dsDNA　probe.　Then　the　AP　sites　will　be　recognized　and　cut　by　endonuclease　(Endo　IV)　to　release　corresponding　single-stranded　primer　probes.　Subsequently,　two　padlock　DNA　templates　are　added　to　initiate　E-RCA　to　generate　multitudinous　G-quadruplexes　and/or　double-stranded　dumbbell　lock　structures,　which　can　combine　N-methyl　mesoporphyrin　IX　(NMM)　and　SYBR　Green　(SGI)　for　the　generation　of　respective　fluorescent　signals.　The　detection　limits　are　obtained　as　low　as　0.0002　U　mL−1　and　0.00001　U　mL−1　for　hAAG　and　UDG,　respectively.　Notably,　this　method　can　realize　the　simultaneous　detection　of　two　DNA　glycosylases　without　the　use　of　specially　labeled　probes.　Finally,　this　method　is　successfully　applied　to　detect　hAAG　and　UDG　activities　in　the　lysates　of　HeLa　cells　and　Endo1617　cells　at　single-cell　level,　and　to　detect　the　inhibitors　of　DNA　glycosylases.　©　2021　Elsevier　B.V.