PRiME HLB purification technique was used to determine the residues of 12 penicillin antibiotics in food by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).In this study, the optimum parameters of purification, concentration, liquid phase separation and tandem mass spectrometry were optimized.The samples were extracted by 80% acetonitrile water mixed solution, purified by PRiME HLB, and separated by CORTECS C18 column (100 mm×2.1 mm, 2.7 μm) with methanol and 0.1% formic acid solution as the mobile phase gradient elution and MRM quantitative analysis.The results showed that each component had a good linear relationship with correlation coefficient R2≥0.998, the average recovery rate was 81.7%-111.9% and the relative standard deviation was 1.82%-8.56%.This method can be used for the determination of penicillin family antibiotics in food.It has the advantages of accuracy, simplicity, sensitivity and high sensitivity.It meets the requirements for the detection of penicillin and antibiotics in China.