The pretreatment method of antibiotic residues in fresh milk was established and optimized. At the same time, 72 kinds of compounds were effectively extracted and purified, and analyzed by ultra performance liquid chromatography-tandem mass spectrometry. In this paper, the effects of different extraction solvents and purification methods on the recovery rate of the target were investigated and the conditions were optimized. 72 antibiotics were linearly good in the range 1.0?Symbol~A@100.0 μg/L. The LOD of the five compounds was 10.0 μg/kg, the LOD of the 15 compounds was 5.0 μg/kg, and the LOD of the remaining 52 compounds was no more than 2.5 μg/kg. The recoveries of 72 kinds of antibiotics were 74%~118.8%, 63.8%~122.6% and 43.9%~139.7%, respectively, at low, medium and high levels, and the relative standard deviations were 1.37%~29.61%, 1.79%~30.27% and 6.03%~39.70%. The method has good reproducibility, short detection time and simple operation, and provides a new rapid detection method for detecting antibiotic residues in fresh milk.