Abstract: In order to investigate the key nodes affecting the changes of Yixian dark tea flavor and quality during the Pile fermentation, in this study, the sensory quality and non-volatile profiles of Yixian dark tea samples at different pile fermentation periods (0, 3, 6, 9, 12, 15, 18 h) were investigated by sensory evaluation, amino acids analyzer, liquid chromatography and liquid chromatography mass spectrometry. The results showed that the intensity of bitterness in dark tea infusions declined after 9 h of pile fermentation, along with the increasing intensities of astringency, thickness and stale-mellow. Total catechins showed a significant decrease in concentrations during pile fermentation. In contrast, the concentration of gallic acid increased. Concentration of caffeine reduced by 14.7% compared to the original concentration after 18 h of pile fermentation. Total amino acids concentration showed an increasing tendency in the later period of pile fermentation. Concentrations of flavonoids and its mono-glycosides increased after 9~12 hours, while its di-glycosides showed an increasing tendency after 12 hours of pile fermentation. The analysis of key taste substances (VIP>1, DOT>1) showed that myricetin-3-O-galactoside, astragalin, isoquercitrin, quercetin-3-O-rutinose, epigallocatechin, epicatechin, epigallocatechin gallate, and caffeine were the most important flavor presenting substances affecting the quality changes of Yixian dark tea. Among them, the content of the first three glycosides increased significantly from 9~12 hours of stacking; The contents of epigallocatechin and epicatechin were significantly reduced during 9~12 hours. Epigallocatechin gallate and caffeine showed a significant decrease after pile fermentation; Quercetin-3-O-rutinoside decreased significantly in the middle stage (9~12 hours) and increased significantly after 15 hours. It was an important factor in the increase of astringency of tea broth in the late stage of pile fermentation. This study give a theoretical basis for improving the tea processing of Yixian dark tea and understanding the formation mechanism of its key flavor.