The low bioavailability exhibits by conventional ocular formulation owing to rapid precorneal clearance and lower corneal permeability can be overcame by the application of the gelling system. In the present study, a prodrug supramolecular hydrogel derived from succinated dexamethasone (Dex-SA) was fabricated using a pH hydrolytic strategy and explored as a “self-delivery” system for ophthalmic drugs. The self-assembled Dex-SA supramolecular hydrogel exhibited a typical nano-fibrous microstructure and was thixotropic. Both dexamethasone (Dex) and Dex-SA prodrug sustainably released from Dex-SA supramolecular hydrogel in a period of 120 h in vitro release study, and the initial pH value of hydrogel significantly influence on the release ratio of Dex/Dex-SA. Furthermore, the lyophilized Dex-SA supramolecular hydrogel displayed long-term stability without causing any apparent hydrolysis of Dex-SA at −20 °C over 30 day and quickly re-formed a hydrogel after dissolving into aqueous solution. The formed Dex-SA supramolecular hydrogel had lower cytotoxicity than Dex at drug concentration up to 2.5 mM, and exhibited a comparable anti-inflammatory efficacy to a Dex sodium phosphate (Dexp) aqueous solution in lipopolysaccharide-activated RAW264.7 macrophages. Topical instillation of the Dex-SA supramolecular hydrogel showed excellent intraocular biocompatibility and it was not an irritant in rabbit eyes. More importantly, the Dex-SA supramolecular hydrogel provided a prolonged precorneal retention and significantly enhanced the ocular bioavailability over Dexp aqueous solution after topical instillation. Overall, this work illustrates an effective approach for the development of prodrug supramolecular hydrogels to extend the precorneal retention and enhance ocular bioavailability of drugs after topical instillation.