The invention discloses a quantitative analysis method of tobacco leaf maleic hydrazide and glucoside of tobacco leaf maleic hydrazide based on gas chromatography-tandem mass spectrometry. The quantitative analysis method comprises the following steps: (1) preprocessing a sample: accurately weighing tobacco leaf sample powder, centrifugal freeze drying, taking out the freeze dried sample, adding pyridine and MSTFA, vortex mixing, standing at a constant temperature, performing silanization for 30 min, obtaining a derivative sample to be detected, and analyzing by a gas chromatography-tandem mass spectrometer; (2) drawing a standard curve; and (3) analyzing the sample: performing the gas chromatography-tandem mass spectrometry analysis for the sample to be detected processed in step (1), obtaining a chromatography-mass spectrometry peak area of the maleic hydrazide and glucoside of tobacco leaf maleic hydrazide in the sample to be detected, inputting into a calibration curve equation, and obtaining, by calculation, the corresponding substance concentration. By adopting the quantitative analysis method, the content of the maleic hydrazide free state and the maleic hydrazide glucosidecombination state in the tobacco leaf can be innovatively detected, the accuracy of the analysis result is greatly improved, and the method is simple and high in efficiency.