Tree peonies are well-known horticultural and medicinal plants. The tree peony seeds, as emerging woody oil crops, recently have attracted great attention for their metabolites and bioactivities. In this study, the phytochemicals isolated from tree peony seed coats were systematically investigated. Seven polyphenolics were separated and prepared, mainly belonging to resveratrol derivatives. There was a great variation in the seed coat polyphenolic content among eight Paeonia species, and the contents of the resveratrol trimers and dimers were significantly higher in the seed coats of Paeonia ostii than other species. Based on the HPLC fingerprint characteristics and chemometric analysis, a clear discrimination among Paeonia plants was found, including the composition patterns and contents of the constituents. Moreover, the characteristic phytochemicals (vateriferol and trans-ε-viniferin) could significantly reduce the starch-mediated levels of postprandial blood glucose in diabetic/normal mice. In addition, in vitro enzyme tests showed that the two compounds could effectively and competitively inhibit α-glucosidase, with the IC50 values of 3.01 and 7.75 μM, respectively, indicating that vateriferol and trans-ε-viniferin could be therapeutic potential agents for hyperglycemia and diabetes mellitus.