G‐quadruplex defines as an evolutionarily conserved sequence, non‐canonical secondary structures found at the telomeric region and in promoter regions of various oncogenes like cMYC, CKIT, and BCL2, etc. Telomerase enzyme activity is inhibited by a small molecule that selectively interacts with G‐quadruplex. Understanding the binding mechanism of the ligand‐G quadruplex complex is crucial for assessing anti‐tumor therapeutic efficacy. We have synthesized different anthraquinone derivative‐based G4 ligands which stabilize quadruplex sequence and influence various cellular processes. We have explored the interaction of synthesized anthraquinone derivatives with G4 DNA sequences comprising human telomeric DNA sequence by using absorption, fluorescence, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR) and thermal melting. Each ligand binds to the G4 DNA in monomeric form. The absorption intensity decreases with a shift of 6‐8 nm. Fluorescence quenching studies and sensorgram indicated a significant binding affinity (Kb) in the order of 102 to 108 M−1. A shift of ~6‐10 nm indicates partial stacking of aromatic chromophores with nucleic acid bases. Similar studies with CD spectroscopy yielded Kb in the range of 103 to 107 M−1. Thermal melting profiling of DNA saturated at Drug/DNA = 5 indicated a stabilization (ΔTm) of 21 to 34 °C in presence of different ions indicating more thermal stabilization in the presence of Na+ ions as compared to K+. Molecular docking analysis concluded that external binding of the ligands to G4 DNAs. The formation of a complex G4 structure was further established by polymerase stop assay. The compounds showed in vitro cytotoxicity in cancer cells (MCF) based on a cell viability assessment which undergoes oxidative lesions at a greater extent. Apoptosis is the mechanical pathway behind the cytotoxicity of cancer cells from the live‐dead staining trials. Modifications of cellular morphs, nuclear condensation, and fragmentation were observed in fluorescence microscopy upon treatment of these compounds. qRT‐PCR highlights gene transcriptional regulation genes containing G4 sequence after treatment of ligands. Apart from damaging G4 DNA, thermal stabilization caused by selective interactions is thought to block telomerase enzyme associated with telomere end and contribute to drug‐induced death in cancerous cell types. The insights expedite therapeutic development due to the extensive possibilities of changing ensuing groups on anthraquinones to improve efficacy, reduce cell toxicity, and increase specificity for G‐quadruplex DNA.