Morus alba leaves is a natural product with great anti-diabetic potential. However, the therapeutic efficacy of natural product 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 enzyme. 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 component in the complex extract from M. alba leaves. As a result, a potential active compound was selectively screened by ligand fishing, separated by HSCCC 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 demonstrated the effectiveness of polydopamine magnetically immobilized bienzyme system for dual-target affinity screening of active substance. This study not only revealed the chemical basis of the antidiabetic activity of M. alba leaves from a dual-target perspective, but also promoted the progress of multi-target affinity screening.