With the growing prevalence of diabetes and obesity worldwide, the role of α-glucosidase and lipase inhibitors has gained increasing concentration. Mulberry, a functional medicinal and edible plant, has been reported extensively in alleviating both diabetes and obesity. However, there is still a lack of refined research on its active ingredients and mechanisms. In our study, the inhibitory activities of mulberry leaf against α-glucosidase and pancreatic lipase reached 191.37 ± 14.46 and 775 ± 68.60 μg/mL, respectively. Meanwhile, it also promoted glucose consumption in the HepG2 cells model. Subsequently, four α-glucosidase ligands and four pancreatic lipase ligands were identified through bio-affinity ultrafiltration combined with liquid-chromatography-mass spectrometry (UF-HPLC–MS/MS). In vitro inhibition assays confirmed the inhibitory potential of these representative ligands. Molecular docking suggested that these possible ligands could be inserted into the active pocket of the target enzyme through multiple intermolecular forces. Our findings indicated that the polyphenols in mulberry leaves may contribute significantly to their hypoglycemic and hypolipidemic properties. Furthermore, our study provides valuable insights for the screening of active ingredients in other medicinal and edible plants, which could help in the development of new therapies for diabetes and obesity.