Glucocorticoids (e.g., dexamethasone (Dex)) are the mainstay in the treatment of non-infectious uveitis, but optimal drug delivery system to maximally realize its potent bioactivity is still lacking. In the present study, we rationally designed and screened a dexamethasone-peptide amphiphile (Dex-GGD), which instantly formed a prodrug supramolecular hydrogel in phosphate-buffered saline (PBS, pH = 7.4) without external stimuli for the topical ocular drug delivery. The obtained Dex-GGD hydrogel was thoroughly characterized by rheology, circular dichroism (CD), and transmission electron microscopy (TEM). The results showed that Dex-GGD exhibited a potent in vitro anti-inflammatory capacity in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages with minimal cytotoxicity against L-929 fibroblasts, ARPE-19 cells, and RAW 264.7 macrophages. In a rat model of experimental autoimmune uveitis (EAU), the severity of uveitis was significantly alleviated after the treatment with Dex-GGD hydrogel owing to its potent ability to reduce the influx of inflammatory cells from peripheral blood and suppress the activation of macroglia and microglia in the retina. In contrast to the treatment with an equivalent dose of dexamethasone sodium phosphate (Dexp), the intravitreal injection of Dex-GGD hydrogel hardly induced the apparent ocular side-effects (e.g., elevated intraocular pressure or fundus blood vessel tortuosity). As a result, the proposed Dex-GGD prodrug supramolecular hydrogel may be an effective and safe therapeutic option for improving the clinical management of uveitis.