Assessing the activity of DNA methyltransferases (MTases) and screening for methyltransferase inhibitors not only allow for a deep exploration of the role of methylation regulation in disease initiation and progression but also provide an important experimental and clinical basis for the diagnosis and treatment of diseases. Herein, a new COFs functionalized electrochemical biosensor has been developed to detect DNA adenine methylation (Dam) MTase activity with high sensitivity and rapidity by taking advantage of the DNA walker and rolled circular strand displacement amplification (RC-SDA) reaction. Specifically, hairpin probe H1 was methylated by Dam MTase, followed by methylation site-specific cleavage of DpnI enzyme to generate the S5 probe. The padlock probes with two nucleic acid endonuclease sites were introduced to trigger the RC-SDA reaction under the action of the primer, releasing a large number of single-stranded S6 probes. The released probe worked synergistically with the Exo III enzyme to trigger DNA walking, which exposed the binding sites to enable the Au-COF-MB signal probes to bind effectively to the electrode surface, and the electrochemical signals were thus generated. The result showed that the designed electrochemical biosensor demonstrated excellent sensitivity and specificity in detecting the activity of Dam MTase, with the detection limit (LOD) of 6.85 × 10