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Sensitive and accurate detection of DNA methyltransferase (MTase) is conducive to the understanding of the fundamental biological processes related to DNA methylation, clinical disease diagnosis, and drug discovery. Herein, a new fluorescence transducer based on Förster resonance energy transfer (FRET) between the donor upconversion nanoparticles (UCNPs) and the efficient acceptor gold nanorods (AuNRs) for MTase activity analysis and its inhibitor screening is presented. A double-strand DNA linker between UCNPs and AuNRs could be digested by restriction endonuclease HhaI, preventing the FRET process and recovering the upconversion luminescence (UCL) intensity. With the treatment of MTase, the cutting site was disturbed by the methylation of cytosine, blocking the enzyme digestion. The transducer presented here showed an excellent analytical performance toward MTase M.HhaI in the concentration range 0.08~24 U mL−1 with a detection limit of 0.057 U mL−1 calculated according to the UCL intensity changes at 656 nm excited by 980 nm CW laser, which is superior to most of the reported methods. Furthermore, the as-fabricated transducer also demonstrated high testing and screening capability toward enzyme inhibitors' evaluation. The method takes the advantage of low background fluorescence of UCNPs to improve the accuracy of the measurement, which can be developed as a general strategy for the analysis of various disease-related methyltransferase activity and their corresponding inhibitors, offering a promising strategy for high-performance diagnosis, high-efficient drug exploitation, and treatment effectiveness evaluation. [ABSTRACT FROM AUTHOR]