Type 2 diabetes (T2D) and its associated complications have emerged as significant global public health challenges. Postbiotics have shown potential benefits in managing T2D. To identify effective postbiotics for T2D amelioration and elucidate the material basis underlying their efficacy, this study developed an anti-hyperglycemic evaluation model. Using an LC-MS platform combined with bioinformatics approaches, we comprehensively analyzed the metabolic distribution profiles of postbiotics in cell-free supernatant (CFS) and cell-free extract (CFE). Through this analysis, we identified a series of notable differential metabolites, including alpha amino acids, D-gulono-1,4-lactone, and glutathione. Spectrum-effect analysis was employed to preliminarily investigate the functional active substances responsible for the anti-hyperglycemic effects of postbiotics, revealing compounds potentially associated with anti-hyperglycemic properties, such as pyroglutamic acid and fumaric acid. Additionally, ultra-filtration mass spectrometry experiments were conducted to target and isolate potential bioactive compounds interacting with α-amylase, α-glucosidase, and tyrosine phosphatase 1B (PTP-1B), including mesoxalic acid, γ-glutamylphenylalanine, and L-3-phenyllactic acid.