Research on the interactions of naturally existing flavonoids with various noncanonical DNA such as i-motif (IM) DNA structures is helpful in comprehending the molecular basis of binding mode as well as providing future direction for the application and invention of novel effective therapeutic drugs. IM DNA structures have been identified as prospective anticancer therapeutic targets, and flavonoids are smaller molecules with a variety of health-promoting attributes, including anticancer activities. The extensive investigation comprising a series of techniques reveals the contrasting mode of the binding behavior of fisetin and morin with various IM DNA structures. We have discovered that structural alterations of hydroxyl groups located at different places of aromatic rings influence flavonoid's reactivity. This minor structural alteration appears to be critical for fisetin and morin's capacity to interact differentially with HRAS1 and HRAS2 IM DNA. Hence, fisetin appears to be an efficient ligand for HRAS1 and morin is considered to be an efficient ligand for HRAS2 IM DNA. This novel exploration opens up the possibility of employing the strategy for regulation of gene expression in cancerous cells. Our finding also reveals the flavonoid-mediated specific interaction with IM DNA while pointing toward tangible strategies for drug discovery and other essential cellular functions.