We report the absolute binding free energy calculation and surface plasmon resonance (SPR) experiment for ligand binding with the cMYC G-quadruplex DNA. The unimolecular parallel DNA G-quadruplex formed in the c-MYC gene promoter regulates the transcription of c-MYC and is recognized as an emerging drug target for developing cancer therapy. Quindoline derivatives stabilize the G-quadruplex and inhibit its expression in cancer cells. NMR experiments revealed two ligand-induced binding sites located at the 5’ and 3’ termini of the c-MYC G-quadruplex. Questions about which site is more favored and the basis for the ligand-induced binding site formation remain unresolved. Here, we employ two absolute binding free energy methods, the double decoupling (DDM) and the potential of mean force (PMF) methods, to dissect the ligand binding specificity in the c-MYC G-quadruplex. The calculated absolute binding free energies ranging from -8 kcal/mol to -11 kcal/mol are in general agreement with the experimental value derived from the SPR measurement (-8.94 kcal/mol). The results suggest that the quindoline compound has a small preference for the 5’-end binding site. Furthermore, the simulations show that the flanking residues involved in the two binding sites undergo significant reorganization as the ligand unbinds, which provides evidence for ligand-induced binding pocket formation. This study provides atomistic insights that help interpret experimental data and inform rational design of small molecules targeting the c-MYC G-quadruplex.