Abstract: A method was established for determining penicillin residues in milk by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and multiwalled carbon nanotubes cleaning. The samples were extracted by acetonitrile and diluted by phosphate buffer solution, a step for clean-up and proconcentration of the analytes by solid phase extraction cartridge packed with multi-walled carbon nanotubes (MWCNTs). The target analytes were separated by Waters C18 column with gradient elution using acetonitrile and 10 mmol/L ammonium acetate (pH 4.5) as mobile phases. The analytes were detected by tandem quadrupole mass spectrometry after positive electrospray ionization by multiple reaction monitoring (MRM). Internal standard method with matrix was used to determine the results. The correlation coefficient is greater than 0.99 for each drugs. The limits of quantitation (LOQ) are 0.1-10.0 μg/kg. The mean recoveries at the three spiked levels are 72.0%-110%. The relative standard deviations (RSDs) are 1.83%-9.33%. The results indicated that using multi-walled carbon nanotubes as solid phase extraction adsorbent is efficient for the purification. The simplicity, sensitivity and good precision of the method made it is well suitable for determination of penicillin residues in milk.