Morus alba leaves are a natural product with great antidiabetic potential. However, the therapeutic efficacy of natural products is usually achieved through the interaction of active compounds with specific targets. Among them, active compounds with multi-target therapeutic functions are more effective than single-target enzymes. In this study, a bienzyme system was constructed by co-immobilizing α-amylase and α-glucosidase onto Fe 3 O 4 for affinity screening of dual-target active components in the complex extract from M. alba leaves. As a result, a potential active compound was selectively screened by ligand fishing, separated by high-speed countercurrent chromatography using a solvent system of ethyl acetate-n-butanol-water (3:2:5, v/v), and identified as rutin. In addition, the result of molecular docking showed that rutin could interact with the active center of α-amylase and α-glucosidase through multiple hydrogen bonds, van der Waals forces, etc. to play an inhibitory role. These results demonstrate the effectiveness of the polydopamine magnetically immobilized bienzyme system for dual-target affinity screening of active substances. This study not only reveals the chemical basis of the antidiabetic activity of M. alba leaves from a dual-target perspective, but also promotes the progress of multitarget affinity screening.