Inspired by the atomic sized, shape-regulated features of G-quadruplexes comprising guanine motifs with a monovalent metal cation, herein we exploited the G-quadruplex-forming ability and properties of a guanine-based π-conjugated Y2 molecule containing bithiophene and peripheral dodecyl chain units in the presence of various cation salts (Li + , Na + , K + , and Mg 2+ ). A series of structural characterization revealed that Y2 yields desirable G-quadruplexes with all the tested cations as a consequence of the combination of a hydrogen-bonded cyclic G-quartet, π–stacking, and cation–dipole interactions. The radius and nature of the coordinating cations crucially affect the structural characteristics of G-quadruplexes, leading to variations in the ion migration ability inside the cavity of the G-quadruplex (Li + > Na + > K + > Mg 2+ ), as characterized through theoretical and experimental investigations. These results not only improve the understanding of G-quadruplex self-assemblies based on guanine but also provide an impetus for their diverse potential applications, especially in the field of Li batteries.