A method for the rapid determination of chloramphenicol(CAP) residues in sea cucumber was developed by isotope dilution liquid chromatography-tandem mass spectrometry. The sample，after addition of aqueous ammonia，was extracted with ethyl acetate. The solution was defatted and purified with n-hexane and high speed centrifugation. The targeted compounds were separated on C18 column（150 mm×2.1 mm，5μm） using water-acetonitrile as mobile phase by gradient elution，and analyzed by a triple quadruple tandem mass spectrometer with electrospray ionization source（ESI）and multiple reaction monitoring（MRM）. The samples were quantified by the internal standards labeled with stable isotopes. The standard curves were linear in the ranges of 0.1-5.0 mg/L for CAP with correlation coefficients（r2）being more than 0.999 2. The limits of detection were 0.06 μg/kg, and the limits of determination were 0.1 μg/kg. The recoveries of CAP in sea cucumber sample at the fortified levels of 0.1-3.0 μg/kg were in the range of 88.2%-98.2%. The intra-assay and inter-assay RSDs for CAP were in the range of 2.3%-4.1% and 3.6%-4.6%，respectively. The results showed that the method was simple，sensitive and accurate，and suitable for the qualitative and quantitative determination of CAP residues in sea cucumber sample.