Effective drug delivery is essential in the treatment of ocular diseases. However, one of the main challenges remains the low bioavailability of drugs at target sites when administered as eye drops, which account for 90% of all ophthalmic treatments. New nanosystem-based delivery methods are needed to maintain adequate drug concentrations over time and improve therapeutic outcomes. To overcome obstacles to topical ocular drug delivery and improve their precorneal retention time for better therapeutic efficacy, gold nanoparticles have emerged as a promising approach to enhance drug bioavailability to the eye. It is therefore crucial to understand the biodistribution profiles of gold nanoparticles in the eye. In this study, in vivo ocular biodistribution is assessed in rabbits up to 24 h after administration of eye drops containing fully characterized polyethylene glycol-modified gold nanoparticles (AuNPs@PEG). The results provide a better understanding of the in vivo biolocalization of ultrastable AuNPs@PEG in various ocular tissues and their potential use as drug delivery systems by demonstrating their in vivo mucoadhesive properties on the ocular surface. A significant amount of AuNPs@PEG is particularly observed in the cornea after gold atom assay by ICP-MS, up to 6 h after exposure to the eye drop, despite the many physical and physiological barriers on the ocular surface. We believe that this AuNPs@PEG accumulation in the cornea can be considered as an advantage for controlling long-term drug release rates. Thus, this residence time of AuNPs@PEG holds promise for improving topically-applied ocular treatments in future drug delivery research.