Highly　sensitive　and　selective　detection　of　DNA　adenine　methylation　methyltransferase　(Dam　MTase)　activity　is　essential　for　clinical　diagnosis　and　treatment　as　Dam　MTase　can　catalyze　DNA　methylation　and　has　a　profound　effect　on　gene　regulation.　In　this　study,　a　fluorescence　biosensor　has　been　developed　for　label-free　detection　of　Dam　MTase　activity　via　methylation-sensitive　cleavage　primers　triggered　hyperbranched　rolling　circle　amplification　(HRCA).　A　hairpin　DNA　probe　(HP)　with　a　Dam　MTase　specific　recognition　sequence　on　the　stem　acting　as　a　substrate　has　been　designed.　This　substrate　probe　can　be　methylated　by　the　target　in　the　system　and　subsequently　cleaved　by　DpnI,　which　results　in　the　release　of　the　primer　release　probe　(RP)　and　hence　in　turn　triggers　the　subsequent　HRCA　reaction.　As　the　HRCA　products　contain　many　double-strand　DNA　(dsDNA)　with　different　lengths,　and　the　SYBR　Green　I　can　be　embedded　in　the　dsDNA　to　produce　a　strong　fluorescence　signal.　However,　in　the　absence　of　the　target,　the　presence　of　the　probe　HP　in　the　form　of　a　hairpin　cannot　induce　the　HRCA　reaction,　and　only　weak　fluorescence　intensity　can　be　detected.　Under　the　optimized　conditions,　the　fluorescence　of　the　system　has　a　linear　relationship　with　the　logarithm　of　the　concentration　of　Dam　MTase　in　the　range　of　2.5–70　U/mL　with　a　detection　limit　of　1.8　U/mL.　The　Dam　MTase　can　be　well　distinguished　from　other　MTase　analogs.　The　developed　sensor　was　applied　to　detect　target　in　serum　and　E.　coli　cell　lysate,　and　the　standard　recovery　rates　were　in　the　range　of　96%–105%.　The　results　showed　that　this　method　has　great　potential　for　assessing　Dam　MTase　activity　in　complex　biological　samples.　In　addition,　the　method　has　been　applied　to　detect　the　related　inhibitors　with　high　efficiency.　©　2020　Elsevier　B.V.