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Abstract We herein developed a novel electrochemical biosensor to detect DNA methylation level, and to quantitatively analyze multiple methylated sites. Graphene oxide was modified with anti-5-methylcytosine antibody to specifically bind CpG methylation sites, and horseradish peroxidase (HRP)-labeled IgG secondary antibody was bound to the former antibody. In buffer containing H 2 O 2 and hydroquinone, HRP-IgG catalyzed the oxidation of hydroquinone into benzoquinone over H 2 O 2 , thereby generating electrochemical reduction signals. The number of 5-methylcytosine was directly proportional to current signal, thereby allowing accurate quantification of methylation level. We also analyzed monomethylated target sequences with different sites. After different methylated sites were captured by the probe, the steric hindrance differences between -CH 3 hydrophobic sphere and the electrode surface were induced. The peak current decreased with reducing distance from the electrode surface, so DNA methylation sites were identified by measuring corresponding peak current responses. With a low detection limit (1 fM), this DNA biosensor was suitable for ultrasensitive DNA methylation detection. The linear detection range was 10–15 M to 10-8 M. Meanwhile, this method had high specificity, stability and repeatability, thus being widely applicable to the clinical detection of DNA methylation. Highlights • A sensitive electrochemical strategy was developed for DNA methylation detection. • Steric hindrance differences from -CH3 hydrophobic sphere to electrode surface identified DNA locus-specific methylation. • Graphene/5-methylcytosine antibody specifically recognized methylated cytosine and improved sensitivity. [ABSTRACT FROM AUTHOR]