Abstract: The aim of the present study was to establish a HPLC method to determine eprinomectin residue in milk. The samples were extracted by acetonitrile and were purified by C-18 solid phase extraction column. After concentration, derivatization and constant volume process, the samples were separated by HPLC and were tested by fluorescence detector. Retention time was used in qualitative analysis and peak area was used in quantitative analysis. The results showed that good linear relationship was obtained in the range of 0.5-100 ng/mL and the correlation coefficients were all above 0. 999; the average recoveries ranged from 65.2% to 96.2% in the adding range of 1-40 ng/g; the inter-assay coefficient of variation was below 15% and the intra-assay coefficient of variation was below 10%. The established HPLC method with high simplicity and sensitivity as well as good stabilization is suitable for the determination of eprinomectin residue in milk.