In this paper, a simple and sensitive method was proposed for 5-methylcytosine determination based on sodium dodecyl sulfate functionalized graphene (SDS-GR) modified pyrolytic graphite electrode (PGE), where SDS-GR was synthesized by electrolyzing graphite rod in 0.01 M SDS solution containing 0.1 M KNO3. After electrochemical activation by cyclic voltammetry (CV), activated SDS-GR modified PGE (A-SDS-GR/PGE) was fabricated for discriminating 5-methylcytosine from other nucleic acid bases. The results indicated that SDS-GR could improve the conductivity of the electrode and promote the electron transfer rate. And the process of electrochemical activation significantly enhanced the amount of active sites and oxygenated groups on electrode surface. As a result, 5-methylcytosine, guanine, adenine and cytosine could be distinguished with different oxidation potentials in a mixture solution except the overlap of the oxidation potential of thymine and 5-methylcytosine. However, the interference caused by thymine could be eliminated because the content of it is equal to adenine. The fabricated A-SDS-GR/PGE provided an alternative route for 5-methylcytosine determination when cytosine existed. (c) 2013 Elsevier Ltd. All rights reserved.