Here, we have explored the light-up property of coumarin 343 (C343) selectively towards various i-motif DNAs based on the recognition of hemi-protonated cytosine-cytosine base pairing, unlike other DNA structures. We have also demonstrated the versatile ability of this i-motif ligand, i.e., the affinity of C343 towards both intermolecular and intramolecular i-motif DNA differing in the chain lengths, molecularity and sizes, which is observed in various oncogene promoters. A systematic study between i-motif DNA and various coumarin derivatives helps in understanding the structural characteristics required for an ideal ligand, which can be useful for future design of any i-motif DNA ligands.