Paeonia ostii is an important economic oil and medicinal crop. Its anthers are often used to make tea in China with beneficial effects on human health. However, the metabolite profiles, as well as potential biological activities of P. ostii anthers and the pollen within anthers have not been systematically analyzed, which hinders the improvement of P. ostii utilization. With comprehensive untargeted metabolomic analysis using UPLC-QTOF-MS, we identified a total of 105 metabolites in anthers and pollen, mainly including phenylpropanoids, polyketides, organic acids, benzenoids, lipids, and organic oxygen compounds. Multivariate statistical analysis revealed the metabolite differences between anthers and pollen, with higher carbohydrates and flavonoids content in pollen and higher phenolic content in anthers. Meanwhile, both anthers and pollen extracts exhibited antioxidant activity, antibacterial activity, α-glucosidase and α-amylase inhibitory activity. In general, the anther stage of S4 showed the highest biological activity among all samples. This study illuminated the metabolites and biological activities of anthers and pollen of P. ostii, which supports the further utilization of them.