Objective To establish a reliable pretreatment method for olaquindox and its metabolite methyl-3-quinoxaline-2-carboxylic acid (MQCA) in milk with ultra performance liquid chromatography-tandem mass spectrometry. Methods The sample was hydrolyzed by hydrochloric acid and extracted by ethyl acetate-acetonitrile (1∶1, V/V), then the concentration was analyzed directly or after PAX and PEP cartridge purification. The objective compounds were separated using Inertsil ODS-3 column (2.1 mm×100 mm, 3 μm) with acetonitrile-water (0.05% ammonia) as mobile phase and analyzed by mass spectrometry in the positive electrospray ionization under multiple reaction monitoring mode(MRM). Results The result showed that direct concentration had good recovery. The detection limit of olaquindox was 0.06 μg/kg, limit of quantitation was 0.20 μg/kg, the average recoveries or spiked levels of 0.20,1.00,5.00 μg/kg were 69.8%,111% and 97.4%. The detection limit of MQCA was 0.02 μg/kg, the limit of quantitation was 0.10 μg/kg,the average recoveries for spiked levels of 0.10,1.00,3.00 μg/kg were 75.8%, 112%, 117%. Conclusion The method was suitable for the detection of olaquindox residual in milk.