OBJECTIVE： To establish a method for the simultaneous determination of chloramphenicol， ephedrine hydrochloride and tetracaine hydrochloride in Dinglyuma eye drop. METHODS：HPLC-DAD at muliti-wavelength was performed on the column of ZORBAX SB-C18 with mobile phase of pH 3.0 Phosphate buffer-acetonitrile （gradient elution）， column temperature was room temperature， flow rate was 1.0 ml/min， injection volume was 20 μl， and the detection wavelength was 277 nm for chloramphenicol， 210 nm for ephedrine hydrochloride and 310 nm for tetracaine hydrochloride. RESULTS： The linear range was 12.56-125.6 μg/ml for chloramphenicol（r＝0.999 8），25.55-255.5 μg/ml（r＝0.999 9） for ephedrine hydrochloride and 25.25-252.5 μg/ml for tetracaine hydrochloride （r＝0.999 9）； RSDs of precision， stability and reproducibility tests were lower than 2％； recoveries were 98.39-100.44％（RSD＝0.79％， n＝9），100.40-101.74％（RSD＝0.66％，n＝9） and 98.83-100.67％（RSD＝0.70％，n＝9）. CONCLUSIONS： The method is sensitive， accurate and specific， and can be used for the quality control of Dinglyuma eye drop.