A　highly　sensitive　electrochemiluminescence　(ECL)　biosensor　for　Dam　methyltransferase　(MTase)　detection　based　on　target-response　DNA　hydrogel　has　been　designed.　The　hydrogel　is　composed　of　hyaluronic　acid　sodium　salt　(HA),　double　strands　DNA　(dsDNA)　that　formed　from　two　amine　modified　oligonucleotides,　a　little　polyethylenimine　(PEI)　and　plenty　of　ECL　signal　probes　(Ru(bpy)32+-doped　SiO2　nanoparticles　(Ru@SiO2　NPs)).　The　dsDNA　has　been　carefully　designed,　which　can　be　recognized　and　methylated　by　Dam　MTase　and　be　specifically　decomposed　by　DpnI　in　turn.　When　target　(Dam　MTase)　existed,　DNA　in　hydrogel　was　destroyed　by　DpnIwhich　results　in　the　leakage　of　signal　probes　into　the　supernatant　and　strong　ECL　signal　can　be　obtained.　Without　target,　DpnI　enzyme　cannot　cut　unmethylated　DNA,　signal　probes　cannot　be　released,　and　low　ECL　signal　can　be　detected　from　the　supernatant.　The　ECL　intensity　detected　has　a　good　linear　relationship　with　the　target　concentration　in　the　range　of　0.05–40U/mL,　and　the　limit　of　detection　is　0.02U/mL.　The　proposed　method　shows　good　selectivity　and　reproducibility,　and　the　prepared　hydrogel　has　good　storage　stability.　The　biosensor　has　been　successfully　applied　for　Dam　MTase　detection　in　human　serum　samples　and　potential　inhibitor　screening　with　satisfactory　results.　©　2021　Elsevier　B.V.