A label-free electrochemical biosensor for methyltransferase (M.SssI MTase) activity detection and inhibitor screening was developed based on the amplification of graphene quantum dot (GQD) and enzyme-catalyzed reaction. In the construction of the electrochemical biosensor, auxiliary DNA hybridized with capture DNA to form the double-stranded DNA structure which contained a specific recognition sequence (5'-CCGG-3') for both M.SssI MTase and restriction endonuclease HpaII. The presence of M.SssI MTase caused the methylation of CpG site in the ds-DNA, which could not be digested by Hpa, causing GQD bound to the undigested ds-DNA and acted as new platform for the immobilization of horseradish peroxidase (HRP) through noncovalant assembly. The modified HRP catalyzed the hydrogen peroxide-mediated oxidation of 3, 3', 5, 5'-tetramethylbenzidine, resulting an electrochemical signal output. The proposed biosensor realized sensitivity detection of M.SssI MTase activity in the range of 1-40 U mL(-1) with a detection limit of 0.3 U mL(-1), and this dual-amplified biosensor had been successfully applied in M.SssI MTase activity inhibitor screening of procaine and epicatechin. Furthermore, this proposed strategy can also be extended for other methyltransferase detection, which had a promising application in clinical diagnosis and drug development.