Many studies have confirmed that DNA methylation is highly correlated with the occurrence and development of various diseases including cancers. In this work, we developed a simple, sensitive, selective, and universal electrochemical biosensor for detection of DNA methylation and assay of DNA methyltransferase (MTase) activity using M.SssI MTase as an example. The thiolated single-stranded DNA SI was self-assembled on the surface of gold nanoparticles deposition modified glassy carbon electrode via Au-S bonding, then hybridization between the DNA S1 and its complementary DNA S2 formed a double-stranded target sequence for both M.SssI MTase and restriction endonuclease HpaII. HpaII could not cleave the target sequence after it was methylated by M.SssI MTase, while the sequence without methylation could be cleaved. Here, we used methylene blue (MB) as electrochemical indicator. The electrochemical signal of MB increased linearly with increasing M.SssI MTase concentration from 0.5 to 25 U/mL and from 25 to 400 U/mL with a detection limit of 0.04 U/mL. Moreover, screening of M.SssI MTase inhibitors 5-azacytidine (5-Aza) and 5-Aza-2'-deoxycytidine (5-Aza-dC) were successfully investigated using the fabricated electrochemical biosensor and showed that the two classic drugs could both inhibit the M.SssI MTase activity with the IC50 of 2.8 mu M and 0.37 mu M, respectively, indicating potential application in discovery of new anticancer drugs.