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I-motif (iM) DNA structures are dynamic cytosine-rich secondary structures that are increasingly recognized for their roles in transcriptional regulation, genomic stability, and for their potential as therapeutic targets in cancer. Despite their significance, the development of selective small-molecule probes for iM DNA remains a challenge. In this study, a series of iminocoumarin-benzothaizole derivatives were designed, synthesized, and subjected to extensive screening to explore their interactions with various iM DNA constructs, including H-Telo, HRAS1, HRAS2, VEGF, and BCL2, as well as duplex DNA. This revealed compounds that display specific and strong interactions with H-Telo, HRAS1, or HRAS2 iM DNA structures depending on their substitution pattern. Detailed spectroscopic investigations revealed the details of how these compounds interact with the iM DNAs, resulting in hypochromic and bathochromic effects, fluorescence enhancements, and increased lifetimes. Furthermore, compounds with unique light-up properties in the presence of HRAS1, VEGF, and BCL2 iM DNA was identified, which has potential as a light-up probes for iM DNA studies in cellular environments. Additionally, circular dichroism (CD) and thermal melting studies confirmed that the compounds stabilized iM DNA without altering its topology, while FT-IR spectroscopy identified structural modifications in iM DNA upon binding. The synthesis of structurally diverse substituents, coupled with extensive spectroscopic, fluorescence, and thermodynamic screening, provided critical insights into structure–activity relationships. Overall, these findings highlight the potential of this compound class to be further developed as selective iM DNA-binding agents and light-up probes, paving the way for innovative diagnostic tools and therapeutic approaches targeting iM DNA in cancer and other diseases.