G-quadruplex, assembled from a square of guanine (G) molecules, is an important structure with crucial biological roles in vivo but also a vesitile template for ordered functional material. Although the understanding of G-quadruplex structures is the focus of numerous studies, little is known regarding the control of G-quartet stacking modes and the spontaneous orientation of G-quadruplex fibrils. Here, the effects of different metal ions and their concentrations on stacking modes of G-quartets are elucidated. Monovalent cations (typically K⁺) facilitate the formation of G-quadruplex hydrogels with both heteropolar and homopolar stacking modes, showing weak mechanical strength. In contrast, divalent metal ions (Ca²⁺, Sr²⁺, and Ba²⁺) at given concentrations can control G-quartet stacking modes and increase the mechanical rigidity of the resulting hydrogels through ionic bridge effects between divalent ions and borate. We show that for Ca²⁺ and Ba²⁺ at suitable concentrations, the assembly of G-quadruplexes results in the establishment of a mesoscopic chirality of the fibrils with a regular left-handed twist. Finally, we report the discovery of nematic tactoids self-assembled from G-quadruplex fibrils characterized by homeotropic fibril alignment with respect to the interface. We use the Frank-Oseen elastic energy and the Rapini-Papoular anisotropic surface energy to rationalize two different configurations of the tactoids. These results deepen our understanding of G-quadruplex structures and G-quadruplex fibrils, paving the way for their use in self-assembly and biomaterials.