Abstract: OBJECTIVE To establish a method for the determination of merthiolate, ethylparaben and benzalkonium bromide in enoxacin eye drops by HPLC, and to investigate the stability of these three preservatives. METHODS The analysis was performed on C₁₈ column, 1% triethylamine (adjusted to pH 3.0 with phosphoric acid) served as mobile phase A, methanol as mobile phase B by gradient elution. The detection wavelength was 218 nm (benzalkonium bromide) and 262 nm (merthiolate and ethylparaben) with the injection volume of 20 μL. The stability of preservatives was investigated. RESULTS The resolution between ethylparaben, merthiolate and benzalkonium bromide was 19.7 and 48.7. The standard curves of merthiolate, ethylparaben and benzalkonium bromide were linear in the range of 9.1-151.4 μg·mL^-1, 2.1-41.2 μg·mL^-1 and 21.8-218.0 μg·mL^-1 (r=0.999, 1.000, 0.999). The limit of detection were 1.4, 1.2, 13.1 ng. The content of merthiolate and ethylparaben decreased lightly under high temperature condition. Merthiolate decreased obviously under UV irradiation condition. Benzalkonium bromide was almost impervious to high temperature and UV irradiation conditions. CONCLUSION The method has high sensitivity and can effectively evaluate the content of preservatives in eye drops.