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.