Tuber melanosporum known as "the black diamond of cuisine", is highly appreciated for its culinary values due to unique and characteristic aroma contributed mainly by its volatile organic sulfur-containing compounds (VOSCs), such as methanethiol (MTL), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), 3-(methylthio)propanal and 3-(methylthio)-1-propanol.In this study, took the aforementioned VOSCs as target product, qualitative and quantitative analysis of the VOSCs in T.melanosporum fermentation system were developed by head-space solid-phase microextraction combination with gas chromatography-mass spectrometry (HS-SPME-GC-MS).L-methionine is considered to be an essential precursor of these VOSCs, then the significance of the addition of L-methionine (i.e., 0, 5, 10, 15 and 20 g/L) on the biosynthesis these VOSCs was studied.Without the addition of L-methionine, the VOSCs were absent.While, with the addition of 5, 10, 15 and 20 g/L of L-methionine, compared with the Demethiolation pathway (i.e., biosynthesis the MTL, DMS, DMDS and DMTS), the Ehrlich pathway (i.e., biosynthesis the 3-(methylthio)propanal and 3-(methylthio)-1-propanol) was major in VOSCs biosynthetic pathway.These results might be very useful to regulate the aroma of T.melanosporum fermentation system.