Since aberrant methylation at CpG sites is linked to the silencing of tumor suppressor genes, DNA methylation analysis is important for cancer diagnosis. We developed ferrocenylnaphthalene diimide (FND), which has two ferrocenyl moieties at the substituent termini, as an electrochemical indicator for hybridized DNA duplexes. In this study, we attempted to detect aberrant methylation of human telomerase reverse transcriptase gene (hTERT), an efficient cancer marker, using FND-based hybridization coupled with electrochemical detection via a multi-electrode chip.