Due to the important role of methylation in cancer, the use of sensitive analytical methods for early diagnosis and efficient clinical pharmacotherapy is highly demanded. In this study, an innovative label-free method has been developed for the recognition of methylated DNA in the promoter area of adenomatous polyposis coli gene (APC gene). Also, differentiation of unmethylated DNA (GCGGAGTGCGGGTCGGGAAGCGGA) from methylated cDNA (GC(M)GGAGTGC(M)GGGTC(M)GGGAAGC(M)GGA) was performed using optical synthesized probe (thionine-based polymer). Hybridization of pDNA (TCCGCTTCCCGACCCGCACTCCGC) with various types of cDNA sequences was studied by UV-visible and fluorescence spectroscopy. Also, some of the mismatch sequences were applied as negative control. For this purpose, The synthesized optical probe was characterized by transmission electron microscopy, atomic force microscopy, dynamic light scattering, zeta potential, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, UV-Vis, and fluorescence spectroscopy. Under optimal conditions, the analytical performance of engineered DNA-based assay was studied and exhibited excellent dynamic range (1 zM to 3 pM) with low limit of quantitation (LLOQ) of 1 zM. The designed DNA-based assay showed a high capability of discriminating methylation, unmethylated and mismatched sequences. The engineered genosensor is simple and inexpensive and can detect DNA methylation with high sensitivity. Therefore, the designed geno-assay could detect DNA methylation significantly and discriminate from unmethylated DNA. It is expected that the proposed geno-assay could be used for the detection of DNA methylation, genetic mutations, epigenetic alterations, and early stage diagnosis of various cancer toward efficient clinical pharmacotherapy.