As emerging woody oil crops, the tree peony seeds recently have been attracting great attention for their metabolites and bioactivities. In this research, the phytochemical profiles of the seed coats of tree peonies from different production regions were investigated systematically. Twelve phytochemicals were separated and prepared, mainly belonging to stilbenes. A great variation in stilbene content was detected in the three Paeonia plants, and Paeonia ostii seed coats (POSC) had significantly higher contents of the stilbene compounds than other species. There were nineteen significant correlations between ecogeographical factors and the predominant compounds. A clear discrimination among the species was observed in their HPLC fingerprint and chemometric analysis. Furthermore, POSC extracts could significantly reduce the starch mediated PBG (postprandial blood glucose) levels in normal/diabetic mice. Meanwhile, in vitro enzyme tests revealed that the predominant compounds, suffruticosol B and ampelopsin D, could effectively and competitively inhibit α-glucosidase, indicating that POSC could be a natural source of hypoglycemics in the food and drug fields.