Abstract A sensitive and simple signal-on electrochemical assay for detection of Dam methyltransferase (MTase) activity based on DNA-functionalized gold nanoparticles (AuNPs) amplification coupled with enzyme-linkage reactions is presented. This new assay takes advantage of the steric hindrance of AuNPs and the electrostatic repulsion between the negative-charge phosphate backbones of DNA modified on the AuNPs and redox probe [Fe(CN)6]3−/4−. In this method, the self-assembled ssDNA on the electrode is hybridized with its complement ssDNA modified on AuNPs to form dsDNA AuNPs bioconjugates containing specific recognition sequence of Dam MTase and methylation-sensitive restriction endonuclease Dpn I. Then, the AuNPs approach to the electrode and result in blockage of electronic transmission. It is eT OFF state. In the presence of Dam MTase and Dpn I, the specific sequence is methylated and cleavaged, which in turn release the DNA modified AuNPs from the electrode surface allowing free exchange of electrons. It generates a measurable electrochemical signal (eT ON). Differential pulse voltammetry (DPV) is employed to detect the recover current, which is related to the concentration of the Dam MTase. This method is simple, sensitive, nonradioactive and without use of gel-electrophoresis, PCR or chromatographic separation. Under optimized conditions, a linear response to concentration of Dam MTase range from 0.2U/mL to 10U/mL and a detection limit of 0.12U/mL are obtained. Furthermore, our new assay is a promising method to detect Dam MTase in the Luria–Bertani (LB) medium, as well as to screen inhibitors or drugs for Dam MTase.