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Hemi-methylated DNA is double stranded (ds)DNA where only one of the complementary strands is methylated. Hemi-methylated DNA levels on a tandem repeat NBL2 and Sat2 are increased in ovarian carcinomas. To quantify hemi-methylation levels, a bisulfite-based method is widely used, but this approach requires multiple steps. Therefore, a simple hemi-methylation assay is required for cancer diagnosis and to elucidate the functions of hemi-methylated DNA. In this study, we aimed to develop a homogeneous assay for the detection of the hemi-methylated DNA level using SET- and RING-associated (SRA) domain-fused luciferase (SRA-luciferase), which recognizes hemi-methylated DNA. In the assay, the hemi-methylation level was quantified by measuring the bioluminescence resonance energy transfer signal between the fusion protein and the DNA intercalating dye. The fusion protein specifically recognizes hemi-methylated DNA to excite the dye by luciferase luminescence. Our results demonstrated that the hemi-methylation level was easily quantified within 35 min with an R2 value of 0.99. [ABSTRACT FROM AUTHOR]