Sensitive and reliable detection of DNA methyltransferase (MTase) is of great significance for both early tumor diagnosis and therapy. In this study, a simple, label-free and sensitive DNA MTase-sensing method was developed on the basis of a nicking endonuclease-mediated multiple primers-like rolling circle amplification (RCA) strategy. In this method, a dumbbell RCA template was prepared by blunt-end ligation of two molecules of hairpin DNA. In addition to the primer-binding sequence, the dumbbell template contained another three important parts: 5′-CCGG-3′ sequences in double-stranded stems, nicking endonuclease recognition sites and C-rich sequences in single-stranded loops. The introduction of 5′-CCGG-3′ sequences allows the dumbbell template to be destroyed by the restriction endonuclease, HpaII, but is not destroyed in the presence of the target MTase—M.SssI MTase. The introduction of nicking endonuclease recognition sites makes the M.SssI MTase-protected dumbbell template-mediated RCA proceed in a multiple primers-like exponential mode, thus providing the RCA with high amplification efficiency. The introduction of C-rich sequences may promote the folding of amplification products into a G-quadruplex structure, which is specifically recognized by the commercially available fluorescent probe thioflavin T. Improved RCA amplification efficiency and specific fluorescent recognition of RCA products provide the M.SssI MTase-sensing platform with high sensitivity. When a dumbbell template containing four nicking endonuclease sites is used, highly specific M.SssI MTase activity detection can be achieved in the range of 0.008–50U/mL with a detection limit as low as 0.0011U/mL. Simple experimental operation and mix-and-detection fluorescent sensing mode ensures that M.SssI MTase quantitation works well in a real-time RCA mode, thus further simplifying the sensing performance and making high throughput detection possible. The proposed MTase-sensing strategy was also demonstrated to be applicable for screening and evaluating the inhibitory activity of MTase inhibitors. •A simple and sensitive DNA methyltransferase (MTase)-sensing method was reported.•High sensitivity is achieved by using a multiple primers-like RCA strategy.•The use of G-quadruplex probe provides a label-free and highly specific mode.•The proposed method can work well in high throughput real-time RCA mode.•The method can also be used for MTase inhibitor screening and evaluation.