To clarify the variation characteristics of flavonoids during the aging process of soybean germplasm resources and their relationship with germplasm vigor, this study used 27 soybean accessions from different origins as experimental materials. Artificial aging treatment at 40°C was applied to obtain different vigor gradients, and the relationships between germplasm vigor indicators, total flavonoid content, and changes in key flavonoid compounds were analyzed. The results showed that: the vigor loss characteristics varied among different accessions; the initial total flavonoid content differed significantly among accessions (0.373-3.381 mg/g), and most accessions showed an upward trend during aging, which was extremely significantly negatively correlated with germination rate. HPLC-MS/MS (High-performance liquid chromatography-tandem mass spectrometry) was used to analyze the flavonoid composition of soybean germplasm with different vigor levels, and combined with principal component analysis, cluster analysis, partial least squares discriminant analysis, and correlation analysis, key differential flavonoid compounds including 7,4'-dihydroxyflavone, calycosin, genistein, and apigenin were identified, which were significantly negatively correlated with germination rate. These key flavonoid compounds were significantly enriched in low-vigor samples and could be used to distinguish soybean germplasm with different vigor levels. Therefore, flavonoid compounds can serve as potential biochemical indicators for evaluating soybean germplasm vigor, providing a theoretical basis for the safe conservation and utilization of germplasm resources.