A method for the determination of clenbuterol，chloramphenicol and diethylstilbestrol in bovine milk was developed by isotope dilution ultra high performance liquid chromatography-tandem mass spectrometry.Samples were directly purified through HLB cartridge with 5 mL water and 5 mL hexane as washing solution.The analytes were eluted with 15 mL ethyl acetate.Then the eluate was evaporated to dryness at 40 ℃ under nitrogen and redissolved with 5 mmol?L-1 of ammonium acetate solution-acetonitrile（90∶10，by volume）.The analytes were analyzed by LC-MS/MS on an Acquity UPLC BEH C18 column using acetonitrile -5 mmol?L-1 ammonium acetate as mobile phase by gradient elution.The mass spectrometer was operated under multiple reaction monitoring（MRM） mode.The samples were quantified by the internal standards labeled with stable isotopes.The calibration curves of clenbuterol，chloramphenicol and diethylstilbestrol were linear in the ranges of 0.2-20，1.2-100,2.0-200 μg?L-1，respectively，with correlation coefficients more than 0.998 5.The limits of detection of clenbuterol，chloramphenicol and diethylstilbestrol were 0.009，0.007,004 ng?g-1，respectively.The recoveries of three drugs in bovine milk sample at the fortified levels of 0.05-0.10（clenbuterol），0.30-0.60（chloramphenicol），0.50-1.0 ng?g-1（diethylstilbestrol） were in the range of 94%-107% with RSDs less than 10%.The method was suitable for the determination of clenbuterol，chloramphenicol and diethylstibestrol residues in bovine milk.