Polygoni Cuspidati Rhizoma et Radix (PCR), the rhizome and root ofPolygonum cuspidatumSieb. et Zucc., has been used as an herbal medicine for a long time. In this study, the ultrafiltration combined with high performance liquid chromatography (UF-HPLC) method was developed to screen tyrosinase (TYR), α-glucosidase (α-GLU), and xanthine oxidase (XOD) inhibitors from PCR. Firstly, the inhibitory activity of 50% methanol PCR extract on TYR, α-GLU, XOD, and acetylcholinesterase (ACHE) was tested. The extract showed a good inhibition on the enzymes, except for ACHE. Therefore, UF-HPLC experiments were carried out to screen TYR, α-GLU, and XOD inhibitors from PCR extract. Seven potential bioactive components were discovered, including methylgallate (1), 1,6-di-O-galloyl-D-glucose (2), polydatin-4′-O-D-glucoside (3), resveratrol-4′-O-D-glucoside (4), polydatin (5), malonyl glucoside resveratrol (6), and resveratrol-5-O-D-glucoside (7). Most of them were found as enzyme inhibitors from PCR for the first time, except polydatin (5), which had been reported as an α-GLUI in PCR in the literature. Finally, molecular docking analysis was applied to validate the interactions of these seven potential active components with the enzymes. Compounds1–7were proven as TYR inhibitors, compounds2,4–7were identified as XOD inhibitors, and compounds4–6were confirmed as α-GLU inhibitors. In short, the current study provides a good reference for the screening of enzyme inhibitors through UF-HPLC, and provides scientific data for future studies of PCR.