Flavonoid represents a significant class of secondary metabolites in Pu-erh tea with benefits to human health. For a rapid and complete discovery of such compounds, we established a data mining workflow that integrates software MS-DIAL, MS-FINDER, and molecular networking analysis. As a result, 181 flavonoids were tentatively annotated including 22 first found in Pu-erh tea, and two of them were potentially new molecules. The dynamic alteration of these flavonoids during Pu-erh fermentation was further investigated. They all showed a trend of first increasing and then decreasing. Moreover, statistical analysis showed that the first to third pile turnings of the fermentation process had a greater impact on the changes of flavonoids. Partial metabolic pathways were proposed. This study provides a quick and automatic strategy for flavonoid profiling. The temporal dimension of flavonoids during fermentation may serve as a theoretical basis for Pu-erh tea manufacturing technology and study on substance foundation.
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