The stability and bioaccessibility of polyphenol from Acacia mearnsii bark were measured at various stages during in vitro simulated digestion. Subsequently, the changes in the total polyphenol content (TPC) and biological activity were studied. The results showed that the phenolic compounds from A. mearnsii remained stable, and TPC underwent few changes during gastric digestion. Nonetheless, intestinal digestion led to the degradation of proanthocyanidins (PAs) and a significant decrease in TPC (26%). Degradation was determined by normal-phase HPLC and gel permeation chromatography. Only monomers, dimers, and trimers of flavan-3-ols were identified in the serum-accessible fraction for characterization of their bioaccessibility. The results also indicated the obvious antioxidant capacity of PAs from A. mearnsii bark, and ~53% of the α-glucosidase⁻inhibitory effect was preserved. All these findings show that PAs from A. mearnsii bark as a native plant source may be particularly beneficial for human health as a natural nutritional supplement.