Abstract: A method for the determination of nicotine and cotinine in fresh edible fungi by gas chromatography-triple quadrupole tandem mass spectrometry was proposed. The sample (5.00 g) was taken, and 5.00 mL of acetone and 25 μL of 2.0 mg·L^-1 internal standard (diphenylamine) solution were added. The above mixture was vortexed for 1 min, and the QuEChERS reagent kit containing 6 g of magnesium sulfate and 1.5 g of sodium acetate was added. After vortex for 1 min and centrifugation for 5 min, the supernatant was filtered through 0.45 μm teflon needle filter, and the filtrate was taken for test. Nicotine and cotinine were separated with column heating program, and mass-to-charge ratio (m/z) 162.10/84.10 and m/z 176.10/98.10 were used as quantification ion pairs, with multiple reaction monitoring mode for scanning and internal standard method for quantification. It was shown that both nicotine and cotinine had matrix enhancement effect, and the linear ranges of working curves were 5.0-200.0 μg·L^-1, with detection limits of 0.5, 2.0 μg·kg^-1, respectively. Test for recovery was made on the blank sample, giving results in the ranges of 97.5%-108% for nicotine and 96.7%-120% for cotinine, and RSDs (n=6) of the determined values were in the ranges of 3.6%-7.7% for nicotine and 2.9%-7.2% for cotinine, respectively. This method was applied to the analysis of 15 fresh edible fungi samples. Nicotine was detected in 13 of them, with detection amounts ranging from 0.000 9 mg·kg^-1 to 0.038 0 mg·kg^-1, and cotinine was not detected.