Objective To establish a method for determination of the content of chloramphenicol in milk powder by liquid chromatography-mass spectrometry, and apply it to test the uniformity and stability of reference material and determine the value. Methods The milk powder was reconstituted by adding water, extracted with ethyl acetate, degreased with n-hexane, extracted with ethyl acetate again, and finally quantified by the isotope internal standard method. Results Chloramphenicol had a good linear relationship in the range of 0.4-4.0 ng/mL, the correlation coefficient was 0.999, the average recovery rate was 100.5%, the relative standard deviation was 1.1%-3.2% (n=6), and this method had a weak matrix effect in milk matrix. Conclusion The method is accurate and reliable, and can be used for the determination of reference materials. After jointly determination by 11 laboratories, the characteristic value and the expanded uncertainty (k=2) of the reference material for the analysis of chloramphenicol residues in milk powder are(1.92±0.30) μg/kg and(5.65±0.53) μg/kg.