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We demonstrate for the first time the integration of a dumbbell probe with dual signal amplification for the simultaneous detection of multiple DNA methyltransferases (MTases). This method is very sensitive with a detection limit of 2.15 × 10−5 U mL−1 for DNA adenine methyltransferase (Dam) and 3.23 × 10−6 U mL−1 for CpG Methyltransferase (M.SssI), and it can be used to screen the enzyme inhibitors and simultaneously measure Dam and M.SssI in complex biological samples. [ABSTRACT FROM AUTHOR]