Abstract: Objective: An analytical method was established for the determination of 16 mycotoxins in traditional Chinese medicines (TCM) by isotope labeling-ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and 483 medicinal and edible homologous samples from the markets were detected using this method. Method: The samples were extracted with acetonitrile-water (50/50, V/V), and then purified by the MycoSpin^TM 400 multifunction clean-up columns. Then the samples were detected and confirmed by UPLC-MS/MS, and quantified by isotope labeled internal standards. Rapid separation of 16 mycotoxins was successfully achieved on an Acquity UPLC BEH C₁₈ column（100 mm×2.1 mm, 1.7 μm）with gradient elution. Simultaneous acquisition was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI) source operated in both positive and negative ionization modes. Result: The established method provided good linearities for the 16 mycotoxins within their respective linear ranges with high correlation coefficients (R>0.998), and the detection limits of this method were 0.1~4.0 μg/kg. The average recoveries of the 16 mycotoxins ranged from 83.4% to 102.3% at the three spiked levels, and the relative standard deviations (RSD, n=6) were in the range of 2.08%~13.6%. In the detection of 483 actual samples, 10 mycotoxins were detected, and the other 6 mycotoxins were not detected. The toxin compound with the highest detection rate was zearalenone (ZEN), with an average content of positive samples 71.2 μg/kg, and 3.11% of the samples exceeded the reference limit specified in the national food safety standard. Conclusion: This method used isotope dilution and multifunction clean-up columns to purify the samples, which reduced the matrix interference in the medicinal and edible homologous samples. The detection limits could meet the requirement of methods. The method was accurate and rapid, and could be used for the detection and analysis of multi residues of mycotoxins in a large number of samples.