A method was developed for determination of amprolium residues in animal origin food by hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry and solid phase extraction (SPE) for sample preparation.Methods The samples were extracted with 1% trichloroacetic acid-acetonitrile (3∶7, V/V). The extract was purified with HLB column. The separation was performed on a SeQuant ZIC-HILIC (150 mm×2.1 mm, 5 μm) column by using 0.1% formic acid-5 mmol/L ammonium acetate solution/acetonitril (40∶60, V/V) as the mobile phase. The analytes was detected by tandem mass spectrometry under multiple reaction monitoring (MRM) mode with positive electrospray ionization (ESI+) and quantified by the matrix-matched external standard method. Results The method showed a good linearity in the range of 0-500 ng/L with the linear relative coefficient above 0.999, the limit of quantification was 10 μg/kg. The average recoveries of spiked levels in chicken meat, chicken liver, chicken kidney, eggs, beef, beef liver and beef kidney, milk and beef fat were 73.2%-101.0% with the relative standard deviations (RSDs) of 2.09%-11.93%.Conclusion This method had good precision, sensitivity, and could be used to quantitative and qualitative detection for the amprolium in animal origin food.