The aroma attributes of sulfurous, mushroom and earthy are the most important characteristics of the aroma of Tuber melanosporum. However, these three aroma attributes are absent in the T. melanosporum fermentation system. To improve the quality of the aroma, repeated freeze-thaw treatment (RFTT) was adopted to affect the interplay of volatile organic compounds (VOCs). Using RFTT, not only was the score on the hedonic scale of the aroma increased from the "liked slightly" to the "liked moderately" grade, but the aroma attributes of sulfurous, mushroom and earthy could also be smelled in the T. melanosporum fermentation system for the first time. A total of 29 VOCs were identified, and 9 compounds were identified as the key discriminative volatiles affected by RFTT. Amino acid analysis revealed that methionine, valine, serine, phenylalanine, isoleucine and threonine were the key substrates associated with the biosynthesis of the 9 key discriminative VOCs. This study noted that amino acid metabolism played an important role in the regulation of the aroma of the T. melanosporum fermentation system.