Uveitis is a sight-threatening complication that continues to be a major contributor to blindness. The etiology of uveitis mostly depends on inflammatory activities. The mainstay of uveitis treatment is the topical use of corticosteroids, although their therapeutic efficiency is constrained by poor corneal penetration and retention. Traditional eye drops are less potent when inflammation extends further into the eye. Nanoemulsions are efficient drug delivery systems for ocular applications owing to their many benefits, particularly sustaining drug action and their capability to penetrate the deepest parts of the ocular structure and the aqueous humor. Herein, a novel preparation of prednisolone-laden cationic nanoemulsion was designed to prolong the precorneal drug retention time, thereby improving the bioavailability of prednisolone for uveitis treatment. Pseudoternary-phase illustrations were created via a water titration approach. A cationic surfactant (cetalkonium chloride) was used to test the effectiveness of a cationic nanoemulsion in extending the precorneal retention of prednisolone. The developed nanoemulsion formulae were assessed for their physicochemical characteristics, morphology, in vitro release profile, and ex vivo permeation patterns. In addition, the clinical investigation and the safety of the proposed formulation in a uveitis-induced experimental animal model were assessed. The proposed nanoemulsion formulations displayed a spherical shape, a nanometer size range, a narrow size distribution, and negative surface charge. The incorporation of cetalkonium chloride decreased the droplet diameter and shifted the droplets' surface charge to positive. The developed cationic nanoemulsions exhibited a sustained in vitro drug release profile and enhanced flux through rabbits' corneas compared to the same formulations without adding cationic surfactant, and free prednisolone suspension (Pred forte® 1 %). Clinical studies showed that using cationic nanoemulsion formulations significantly reduced the severity of uveitis in rabbits' eyes throughout treatment period (three weeks) compared to drug suspension (Pred forte® 1 %). Prednisolone cationic nanoemulsion formulations did not cause an elevation in intraocular pressure (IOP) and any appreciable changes in the diameter of the rabbits' pupils in the investigated animal groups. Also, there were no adverse effects on the cornea, retina/choroid, or iris/ciliary body, demonstrating the safety of the suggested nanoemulsion formulations. Therefore, the developed prednisolone cationic nanoemulsion system may offer a potential vehicle for ophthalmic drug delivery and enhanced management of uveitis.