A method based on ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/MS) was developed for simple and rapid determination of the residues of Auramine O in animal feedstuffs. The samples were extracted by MeOH: H2O + HCOOH 0.1% and then analyzed in multiple reaction monitoring (MRM) mode. The mobile phase was ultrapure water with 0.1% formic acid. Under the optimized detection conditions, the linear range for Auramine O was 20 - 100µg/L and the linear correlation coefficients found more than 0.99. The limit of quantification of Auramine O was 0.34 mg/kg. The recoveries of Auramine O ranged from 64.71 - 94.12% with relative standard deviations (RSD) of 4.93 - 8.31% with the concentration range of 20 - 100 µg/L. This method is simple, effective, sensitive and is suitable for the determination and confirmation of Auramine O in animal feedstuffs.