In　this　work,　an　electrochemiluminescence　(ECL)　biosensor　with　dual　signal　enhancement　was　constructed　and　used　for　DNA　adenine　methylation　methyltransferase　(Dam　MTase)　detection.　At　present　of　Dam　MTase,　restriction　endonuclease　(DPnI)　cleaves　hairpin　DNA　(HP)　and　releases　the　HP　stem　end　as　a　single　strand　that　can　activate　CRISPR/Cas12a　trans-cleavage　activity.　Assisted　by　trans-cleavage,　the　distance　between　the　signal　quenching　factor　ferrocene　(Fc)　and　the　ECL　signal　unit　increased,　and　the　repulsion　between　the　signal　unit　and　the　Indium　Tin　Oxides　(ITO)　electrode　decreased.　The　above　results　resulted　in　an　enhanced　ECL　signal.　ECL　intensity　has　a　good　linear　relationship　with　the　logarithm　of　Dam　MTase　concentration　in　the　range　of　5–70　U/mL　with　a　detection　limit　of　23.4　mU/mL.　The　proposed　biosensor　was　successfully　utilized　to　detect　of　Dam　MTase　in　serum　samples.　©　2022　Elsevier　B.V.