Objective To observe the efficacy of levofloxacin hydrochloride eye gel (LHEG) in treatment of Staphylococcus aureus keratitis in rabbits. Methods Sixty rabbits with normal eyes were used in this study; their left eyes received intrastromal injection of 10 μL bacterial suspension containing 10⁵ colony-forming unit (CFU) Staphylococcus aureus to establish Staphylococcus aureus keratitis model. Forty-eight rabbits with similar severity of inflammation were randomly divided into four groups:24 h baseline group, phosphate buffer solution (PBS) control group, levofloxacin hydrochloride eye drop (LHED) group, and LHEG group. Rabbits in the 24 h baseline group received slit-lamp examination and were given clinical score, and their corneas were collected for histopathology examination and bacterial quantitation. The other three groups received treatment with PBS, LHED or LHEG three times a day for seven successive days. In the last day, rabbits in these three groups received the same examination as that in the 24 h baseline group. The clinical scores and cornea bacterial quantitation were analyzed with variance analysis, and a P value less than 0.05 was considered as statistically significant. Results Compared with PBS control group, clinical symptoms of LHED group and LHEG group were significantly reduced(P=0.00) on the seventh day, with the clinical scores being (4.21±1.10) and (3.63±0.86), respectively; the numbers of bacterial colonies were also significantly reduced in LHED group and LHEG group (P=0.00), with the numbers being (4.87±0.05) and (4.64±0.10) CFU, respectively. Moreover, LHEG group had a significantly better bacteriostatic effect than LHEG group (P=0.00). Conclusion Local application of LHEG can greatly inhibit the growth of bacterial colony of Staphylococcus aureus and improve the clinical signs of keratitis, with the efficacy being better than that of routinely used LHED in rabbit model of Staphylococcus aureus keratitis.