DNA methylation is critical for biological processes in prokaryotes and eukaryotes; its aberrations are tightly linked to the onset and progression of numerous diseases. As a key epigenetic component, it also sustains normal human cell function, regulates gene imprinting, and participates in oncogenesis. Clustered regularly interspaced short palindromic repeat (CRISPR)-based systems (e.g., CRISPR-Cas12a) are programmable and sensitive, making them valuable for biosensors—Cas12a exhibits unique trans-cleavage activity, randomly cleaving ambient single-stranded DNA (ssDNA) upon activation by target DNA. On the basis of this, we developed a sensitive assay for DNA methyltransferase (MTase) activity using the CRISPR-Cas system, employing a DNA tetrahedron with four dumbbell-ring-containing corners. In this assay, Dam MTase modifies the N6 position of adenine in the GATC sequence; the modified sequence is then cleaved by DpnⅠ restriction endonuclease and associates with CRISPR-Cas12a. The exposed ssDNA activates Cas12a, which cleaves a fluorescent ssDNA probe to generate a signal—Dam MTase concentration is quantified via fluorescence intensity. This method detects Dam MTase in human serum, screens its inhibitor 5-fluorouracil (5-FU), and shows high sensitivity/specificity [linear range 0.01–1 U/mL (R2 = 0.99715), limit of detection 0.001 U/mL]. It holds promise for early cancer diagnosis and clinical applications, representing a valuable research direction for related studies.