DNA　methyltransferase　(DNA　MTase)　can　act　as　biomarker　for　many　diseases　and　it　is　important　to　develop　some　new　methods　for　sensitive　detection　of　DNA　MTase.　In　this　work,　a　highly　efficient　electrochemiluminescence　(ECL)　sensor　had　been　designed　for　detection　of　DNA　MTase　based　on　Ru(phen)(3)(2+)　loaded　double　strand　DNA　(dsDNA-　Ru(phen)(3)(2+))　as　signal　tags.　Ru(phen)(3)(2+)　had　been　efficiently　embed　in　the　dsDNA　produced　through　a　simple　hybridization　chain　reaction.　First,　a　hairpin　probe　was　designed,　which　can　be　specifically　recognized　by　Dam　MTase　and　modified　with　-SH　at　one　end.　It　was　modified　on　the　surface　of　gold　electrode　by　-SH　as　an　immobilization　probe　(IP).　This　IP　will　be　methylated　in　the　present　of　Dam　MTase　and　digested　by　DpnI　following.　Results　in　the　release　of　capture　probe　(CP)　which　remains　on　the　surface　of　gold　electrode.　The　CP　can　hybridize　with　the　single　stand　part　of　the　dsDNA-　Ru(phen)(3)(2+)　and　make　the　immobilization　of　ECL　tags　on　the　electrode　surface,　which　results　in　a　strong　ECL　signals　detected.　However,　without　the　effect　of　Dam　MTase,　the　hairpin　structure　of　IP　remains　stable　and　cannot　capture　signal　tags,　and　can　only　detecte　weak　ECL　signals.　The　biosensor　can　detect　the　activity　of　Dam　MTase　in　the　concentration　range　of　0.01　U/mL　to　20　U/mL　with　the　ECL　intensity　and　the　logarithm　of　the　concentration　have　a　linear　relationship,　and　the　detection　limit　is　calculated　to　be　7.6　mU/mL.　The　developed　sensor　has　the　ability　to　specifically　detect　Dam　MTase,　which　can　be　differentiated　from　other　types　of　DNA　MTase.　In　addition,　the　designed　method　has　good　applicability　to　detect　Dam　MTase　activity　in　serum　samples　and　been　applied　to　detect　its　inhibitor　with　high　efficiency.