DNA methylation has been proposed as one of the promising biomarkers for cancer diagnosis. In this study, we developed a DNA methylation detection system utilizing G-quadruplex and i-motif-forming sequences that requires neither sodium bisulfite treatment nor methylated DNA ligands. We hypothesized that G-quadruplex and i-motif structures would be stabilized by DNA methylation and arrest DNA polymerase activity during quantitative polymerase chain reaction (qPCR). The PCR products from VEGF, RET G-quadruplex, and i-motif-forming sequences were used as templates and analyzed by qPCR. Our results indicated that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels in the G-quadruplex and i-motif-forming sequences. Moreover, we demonstrated that the initial elongation efficiency of PCR decreased with increased DNA methylation of the VEGF region on genomic DNA. These results indicated that DNA methylation of the G-quadruplex and i-motif-forming sequences on genomic DNA can be detected by qPCR.