Abstract: To simultaneously enrich polyphenols and β-glucan in Lactobacillus-fermented oats and enhance their activity in regulating glucose and lipid metabolism, single-factor experiments and response surface methodology (RSM) were employed to optimize fermentation conditions. Untargeted metabolomics was utilized to analyze the composition and relative abundance of polyphenols in fermented oats. Subsequently, the antioxidant activities of aqueous and ethanolic extracts from fermented oats, as well as their inhibitory effects on key enzymes involved in glucose and lipid metabolism, were evaluated. Furthermore, representative polyphenolic components identified in fermented oats were used as ligands for molecular docking to elucidate the molecular mechanisms underlying the inhibition of these key enzymes. The results demonstrated that fermentation with Lactobacillus kisonensis (JCM 15041) under optimal conditions (inoculum size: 2%, solid-to-liquid ratio: 1:6 g/mL, fermentation time: 6 h, temperature: 30 ℃) significantly increased the β-glucan content in oats by 61.54%, while enhancing the inhibition rates against α-amylase and α-glucosidase by 28.61% and 24.11%. Compared to unfermented oats, both the ethanolic and aqueous extracts of fermented oats exhibited significantly enhanced antioxidant activity (P<0.05) and inhibitory effects on key glucose and lipid metabolism enzymes (P<0.001). Untargeted metabolomics revealed a significant increase (P<0.01) in the abundance of cinnamic acid post-fermentation. Molecular docking using cinnamic acid as the ligand indicated its potential binding modes: forming two hydrogen bonds (2.1 Å) with Lys-466 of α-amylase (binding energy: −4.59 kcal/mol); two hydrogen bonds (2.2 Å) with Phe-416 and Lys-418 of α-glucosidase (binding energy: −4.43 kcal/mol); and five hydrogen bonds (1.6 Å, 2.1 Å, 2.4 Å, 2.6 Å, and 2.8 Å) with Pro-430, Arg-457, and Glu-458 of pancreatic lipase (binding energy: −5.77 kcal/mol). These interactions suggested that cinnamic acid likely inhibits enzyme activity by affecting the substrate-binding domain and catalytic active site. In conclusion, this study established a Lactobacillus-fermentation process capable of significantly enriching oat β-glucan. The resulting fermented oat extracts possessed potent in vitro antioxidant activity and significant potential for regulating glucose and lipid metabolism.