Objective:To establish a method for the determination of chloramphenicol and hydrocortisone in chloramphenicol hy-drocortisone ear drop. Methods:An HPLC method was used with a Shiseido SPOLAR C18 column (250 mm ′4. 6 mm, 5 mm). The mobile phase was 0. 01 mol·L-1 sodium heptane sulfonate buffer solution (6. 8 g potassium dihydrogen phosphate was dissolved in 0. 01 mol · L-1 heptane sodium sulfonate buffer solution and diluted to 1000 ml, 5 ml triethylamine was added and mixed, and then the pH was adjusted to 2. 5 by phosphoric acid)-methanol (40︰60). The column temperature was 30℃ and the flow rate was 1. 0 ml · min-1 . The detection wavelength was 245 nm and the injection volume was 10 μl. Results: Chloramphenicol and hydrocortisone had a good linear relationship within the range of 50. 26-753. 84 μg · ml-1 ( r =0. 9996 ) and 10. 93-163. 92 μg · ml-1 ( r =1. 0000), respectively. The average recovery of chloramphenicol was 100. 21% and RSD was 0. 48%(n=9). The average recovery of hydrocortisone was 100. 82% and RSD was 0. 37% (n=9). Conclusion:The method has the advantages of high specificity, good reproducibility and high precision, and can be used as a method for the determination of chloramphenicol and hydrocortisone in chlor-amphenicol hydrocortisone ear drop.