To establish an HPLC method for the determination of levofloxacin hydrochloride in levofloxacin hydrochloride ophthalmic gel. The anal. was performed on an C18 column (250 mm×4.6 mm, 5μm) and the mobile phase was acetonitrile-buffer solution (15: 85) that the buffer solution was 4.0 g ammonium acetate and 7.0 g sodium perchlorate dissolved in 1300 mL water, then adjusted to pH 2.2 with phosphoric acid. The flow rate was 1.0 mL·min-1,the UV wavelength was 294 nm, and the column temperature was at 25°C. The detection concentration of levofloxacin hydrochloride shoed a fine linear relation with peak areas between 60∼160 μg·mL-1 (r=0.9999). In the recovery test, the average recoveries and RSD of levofloxacin hydrochloride were 101.22% (0.30%), 101.01% (1.30%), 100.49% (1.28%) resp., which indicated preferable precision and accurate. The results of three batch samples of HPLC closed to national standard method, therefore it is appropriate to be used in the quality control of the levofloxacin hydrochloride ophthalmic gel.