High-throughput sequencing was used to analyze the fungi diversity and dominant genera in Yixian dark teapile, and gas chromatography mass spectrometry (GC-MS), liquid chromatography (LC) and amino acid analyzer (AAC) were used to analyze the changes of volatile compounds and major taste substances in the samples before and after pile-fermentation.The results showed that the temperature of Yixian dark tea was the highest at the beginning of pile-fermentation, and then the temperature between the layers gradually decreased. The diversity of fungi was low at the beginning of pile-fermentation, and was the richest in the middle period (8-12h), and the diversity of the upper layer of the pile-fermentation was higher than that of the lower layer, and the lower layer is higher than the middle layer. A total of nine dominant fungi including Didymella, Cladosporium, Setophoma, Alternaria, Bulleromyces, Zymoseptoria, Fusarium, Strelitziana and Penicillium were detected in pile-fermentation, with the Didymella, Cladosporium, Setophoma, Alternaria and Bulleromyces accounted for more than 50% of the total number of fungi in pile-fermentation.The study of volatile components and main taste substances of Yixian dark tea before and after pile-fermentation found that the total amount of volatile substances increased significantly after pile-fermentation, among which the relative content of glycosidic aromatic substances, such as dihydrolinalool, linalool, geraniol, lauric acid, methyl salicylate, and etc., increased significantly, which was a key factor influencing the change of aroma. The effect of pile-fermentation on the total amount of free amino acids and caffeine content was small, but the effect on catechin substances was big, and its total amount decreased by 13%, which was mainly decreased in ester-type catechin substances. The research explored the fungi diversity in Yixian dark tea pile-fermentation and the influence of pile-fermentation on quality formation, which provided a theoretical basis for the processing and quality formation of Yixian dark tea.