We demonstrated the robust and fully reversible photoisomerization of the diazocine ligand under high cationic conditions and show a >50-fold difference between the E and Z isomers as determined by UV/Vis and CD titration studies. CD experiments further demonstrated that the ligand binding and dissociation from the LTR-III G4 can be controlled reversibly by alternate irradiation with blue light (λ = 405 nm) and green light (λ = 520 nm). Further evidence of the differential behaviour between the E-and Z-isomers was obtained by 1 H-NMR studies in combination with molecular dynamic calculations. These experiments suggest the (E)-ligand binds preferentially at the G4/duplex junction of the LTR-III sequence, whereas the (Z)-isomer favors the duplex region. Our results demonstrate that the conformational switch of the ligand by an external stimuli such as light can be used to control binding affinity and mode of binding in a fully reversible manner, without the need to pre-incorporate photoresponsive functionality in the biomolecule. Further investigations into the activity and application of these type of G4 ligands are already underway and will be reported in due course.