Abstract: A method for simultaneous determination of aflatoxins B₁, B₂, G₁, and G₂ in pheretima was been established by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and the positive results was confirmed. The samples were extracted by 70% methanol, purified by a immunoaffinity columns, separated by UPLC-MS/MS, detected by triple quadrupole mass spectrometry, and confirmed by ion peak ratio. The results showed that the linear relationship of the four aflatoxins were good (r>0.999 5), and the recoveries ranged from 91.2% to 95.6%. The limits of detection for aflatoxin B₁, B₂, G₁, and G₂ were 0.10, 0.038, 0.11, 0.038 μg/kg, respectively. The method has a good specificity, high sensitivity, good accuracy, and can be used to determine the final positive detection. Aflatoxin were positively detected in 6 out of 15 batches of pheretima, which proved that the Pheretima were more susceptible to contamination by aflatoxins.