This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification(RCA) technique. In the presence of DNA adenine methylation(Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons(detection probes), resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis. This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification (RCA) technique. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The products cleaved by restriction endonuclease Dpn I then function as signal signal primer to hybridization with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of The proposed method exhibits a dynamic range from 0.5 U / mL (detection probes), resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation / cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. to 30 U / mL and a detection limit of 0.18 U / mL. This method can be used for the screening of antimicro bial drugs and has a great potential to be further applied in early clinical diagnosis.