DNA methylation is a major epigenetic modification that is closely related to human health. Many experimental techniques as well as theoretical methods have been used to detect the modified nucleotides and identify their effects on molecular binding. It remains challenging to resolve the effect of few methylations of nucleic acids. Using super-resolution force spectroscopy, we firstly revealed that single cytosine methylation increases the mechanical stability of the DNA duplex by 1.9 ± 0.3 pN. Methylation also induces significant chiral selectivity towards drug molecules such as d,l-tetrahydropalmatine. Our results precisely quantify the mechanical effect of methylation and suggest that drug design should take methylation into consideration for enhanced selectivity.