Abstract: Timolol maleate cubic nanoparticles (TM-LCNPs) were prepared via fragmentation of a bulk GMO/poloxamer 407 cubic phase gel by high-pressure homogenization. The optimal prescription was selected based on particle size and entrapment efficiency by orthogonal design method. Malvern particle sizer, polarized light microscopy, and differential scanning calorimetry were used to characterize the cubic nanoparticles. Commercial eye drops were used as a control for the release and corneal permeation experiment in vitro. Fluorescence imaging was used to observe the retention of Rhodamine B cubic nanoparticles (RhB-LCNPs) in rabbit cornea. The results indicated that the optimal prescription and preparation of TM-LCNPs was oil-water ratio (7:3), homogenous pressure (900 bar), the number of homogenizations (6) and drug loading (1%). Corneal permeability of TM-LCNPs was significantly higher than that of commercially available eye drops. The residence time in eyes was longer which suggested a sustained release behavior. The pathology result of rabbit corneal after multiple administration of TM-LCNPs showed that there was no apparent damage.