Supramolecular hydrogels derived from natural biomolecules have promising applications for drug delivery due to their inherent biocompatibility and tunable responsiveness to various stimuli. However, conventional hydrogels only modulate the release kinetics roughly to achieve sustained drug release, exhibiting fast-then-slow release behavior without on/off control. Herein, a guanosine (G)-quartet·Na⁺-borate supramolecular hydrogel (GB hydrogel) cross-linked via a guanosine–borate diester and intertwined by G₄-nanofibres formed by π–π stacking of G₄-quartets stabilized by Na⁺ is developed for on-demand release of Acyclovir (Acv). This GB hydrogel is facilely prepared by a one-pot hierarchical assembly involving hydrogen bonds, dynamic borate ester bonds and cation coordination, which endow it with tunable mechanical properties, excellent self-healing properties and reversible degradation behavior in response to pH, glucose and ion concentration. Benefiting from that the guanosine analog Acv is able to assemble into a G₄-quartet by replacing guanosine via reversible hydrogen bonding, the Acv-loaded GB hydrogel showed favorable stability in physiological medium without undesired release and achieved external stimulus-triggered on-demand release with switchable on/off control and tunable release kinetics. Moreover, the GB hydrogel also exhibited excellent in vitro and in vivo biocompatibility. Such a natural nucleoside-based supramolecular hydrogel with on-demand drug release, self-healing property, biodegradability and biocompatibility provides a precisely controlled paradigm to overcome early burst release behavior of conventional hydrogels for the development of injectable hydrogel delivery systems.