A accurate and quantitative method was developed for the determination of vancomycin antibiotics residues in animal food by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The samples were extracted by formic acid acetonitrile solution, degreased by acetonitrile-saturated n-hexane, purified by cation solid-phase extraction column, detected in the multi reaction monitoring (MRM) positive ion mode, and quantified by internal standard method using didechloro vancomycin as internal standard. The results showed that the target of vancomycin and norvancomycin had a good linear relationship in the range of 20~200 μg/L, and the correlation coefficient (R²) could reach more than 0.997. The average recoveries varied from 88.7% to 115.1% at the spiked levels of 20, 50 and 200 μg/kg, and the relative standard deviations were in the range of 3.1%~9.4%. The limit of detection (LOD) for the method was as low as 1.0 μg/kg, and limit of quantitation (LOQ) could reach 3.0 μg/kg. This method is rapid, sensitive, accurate, reproducible and suitable for the determination of vancomycin antibiotics residues in animal food.