Liquid chromatography coupled with tandem mass spectrometry ( LC-MS /MS) method was established for simultaneous determination of ergosterol and vitamin D2 content in Lentinus edodes． Ergosterol and vitamin D2 were extracted by saponification reflux combined in ultrasonic auxiliary and purified through C8 chromatographic column with optimizing the chromatographic conditions at 25 ℃ of column temperature，270 nm of detection wavelength and 1. 5 mL /min of the flow rate of methanol aqueous solution in the gradient elution methods． The prepared samples were tested by mass spectrometry with electrospray ionization positive ion ( ESI + ) mode and multi-reaction ( MＲM) monitoring mode． Ion source injection voltage ( IS) at 5500V，ionic source temperature ( TEM) at 500℃ and ion source spray velocity at 5 L /min were applied as mass spectrometer conditions when the set pressure of air curtain gas ( CUＲ) ，atomization gas ( GS1 ) and auxiliary gas ( GS2 ) were controlled at 137. 9，310. 3 and 413. 7 kPa， respectively． Ｒesults show that the retention time of ergosterol and vitamin D2 chromatographic peaks are at 4. 49 and 4. 26 min，respectively，the content of ergosterol ( 0. 40 ～ 3. 60 mg /L) and vitamin D2 ( 0. 22 ～ 2. 20 mg /L) within a certain content range of peak area presents a good linear relationship． The average recovery rate of ergosterol and vitamin D2 are within 86. 7% ～ 102. 4% ，and the relative standard deviation are within 1. 02% ～ 4. 13% ，respectively，according to methodological validation． The collection samples were detected by LC-MS / MS established in this experiment and HPLC recommended by the national standard． The detecting results suggest that no significant difference ( P ＞ 0. 05) is found by two methods utilized for samples analysis． Compared with the peak area quantification by HPLC chromatography，LC-MS /MS method has the advantages of shorter detection time，lower detection limit and more sensitive detection． The simultaneous determination of ergosterol and vitamin D2 in Lentinus edodes by liquid chromatography-tandem mass spectrometry established in this study lays a foundation for the establishment of new product quality standards and product quality testing standards for edible fungi，and provides theoretical basis for the simultaneous detection and research of isomers．