In this study, a ligand fishing technique based on magnetic mesoporous silicon was established and used to screen α-glucosidase inhibitors from Pueraria lobata. To clarify quantity-activity relationships in a holistic view, the knock-out/knock-in technology was used to analyse the interactions of several active constituents in P. lobata. Magnetic mesoporous silicon with a large specific surface area and better biocompatibility was synthesised. Subsequently, α-glucosidase was immobilised on -NH2-modified magnetic mesoporous silicon, and the compounds in the crude extract of P. lobata were screened across enzyme binding. The structures of the ligands were elucidated using UPLC-Q-TOF-MS/MS, and their activities were verified by knock-out/knock-in experiments and molecular docking. Daidzein and puerarin showed α-glucosidase inhibitory activities with an IC50 of 0.088 ± 0.003 mg mL-1 and 0.414 ± 0.005 mg mL-1, respectively. Among them, puerarin, which accounted for more than 40% of the total content, showed synergistic effects with other components and was the main contributor to the α-glucosidase inhibitory activity of P. lobata.