Rutin, a naturally occurring flavonoid, exhibits potent antioxidant and anti-inflammatory properties, making it a promising therapeutic candidate for various ocular disorders. However, its clinical applications are hindered by poor aqueous solubility and low ocular bioavailability. This study aimed to develop and optimize nanostructured lipid carriers (NLCs) to encapsulate rutin and enhance its ocular delivery. Rutin-loaded NLCs (RT-NLCs) were prepared by a melt-emulsification and ultrasonication technique using varying concentrations of rutin (0.05-0.5% w/v). The formulations were characterized for particle size, polydispersity index (PDI), entrapment efficiency, and transcorneal permeability. Based on these parameters, the RT-NLC formulation containing 0.1% w/v rutin (F3) was identified as the optimized formulation, exhibiting a mean particle size of 229.50 ± 50.58 nm, PDI of 0.454 ± 0.083, and entrapment efficiency of 69.36 ± 0.54%. Transmission electron microscopy revealed the spherical morphology and smooth surface of the optimized RT-NLCs. In vitro release studies demonstrated controlled release of rutin from the optimized formulation, following the Higuchi kinetic model. Significantly, ex vivo transcorneal permeation studies using excised goat cornea showed a 6-fold higher permeability coefficient (134.99 ± 36.21 cm/s) for the optimized RT-NLC compared to F5 rutin NLC formulations. Furthermore, the hen's egg test-chorioallantoic membrane (HET-CAM) assay confirmed the non-irritant nature of the optimized formulation. In conclusion, the nanostructured lipid carrier system demonstrated promising potential for improving the ocular bioavailability of the poorly soluble drug rutin.