Abstract: To investigate the digestion characteristics of theaflavins (TFs) and their effects on oral and gut microbiota, this study simulated gastrointestinal digestion in vitro, measuring changes in TFs content and antioxidant activity during digestion, and determined alterations in the composition of oral and gut microbial communities via 16S rRNA high-throughput sequencing. The results showed that during simulated oral digestion, the total TFs content decreased, and antioxidant capacity significantly weakened with prolonged digestion time (P<0.05). The stability order of the four monomers was TF2B>TF1>TF3>TF2A. During simulated gastric digestion, the total TFs content decreased compared to before digestion but increased relative to the oral digestion stage, while antioxidant capacity declined significantly compared to pre-digestion levels (P<0.05). The stability order of the four monomers remained TF2B>TF1>TF3>TF2A. In simulated intestinal digestion, the total TFs content showed no significant changes, with antioxidant capacity significantly reduced (P<0.05). The stability order of the monomers shifted to TF2B>TF1>TF2A>TF3. Oral microbiota analysis indicated that TFs significantly inhibited the growth of pathogenic bacteria such as Haemophilus, Veillonella, and Porphyromonas, while promoting the proliferation of probiotics like Lactiplantibacillus. Gut microbiota studies revealed that TFs selectively modulated microbial structure, enhancing the growth of beneficial bacteria such as Megamonas and Bacteroides, which produce short-chain fatty acids, and suppressing the overgrowth of conditional pathogens like Escherichia-Shigella. These findings provide theoretical support for improving the bioavailability of TFs and expanding their applications in the food industry.