Abstract: Aflatoxin, affecting the quality and safety of Pheretima, is one of the major risk factors. The QuEChERS-dispersed solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (QuEChERS-dSPE-UPLC-MS/MS) has been established for the rapid determination of aflatoxin in Pheretima powder. The sample was extracted with quick, easy, cheap, effective, rugged and safe (QuEChERS), then purified with the enhanced matrix removal lipid-dispersed solid phase extraction (EMR-Lipid-dSPE). The multiple reaction monitor (MRM) acquisition mode was used for qualitative and quantitative detections, and an external standard method was used for the quantitative detection. The analytes showed good linearities in corresponding concentration ranges of 0.00~1.60 ng/mL with their correlation coefficients (R²) between 0.990 0 and 0.994 1. The limit of detection were 0.013~0.365 ng/g, the limit of quantitative were 0.044~0.913 ng/g, the spiked recoveries for analytes at three spiked levels were in the range of 72.0%~113.0%, the precision were 2.5%~3.3%. No aflatoxin was detected in 10 batches of samples. The matrix removal effect of this method is sufficient, the pretreatment method is simple, reproducible and sensitive, and is suitable for the rapid screening of aflatoxin in Pheretima.