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Morus alba L has long been used as fodder and as a traditional medicine. The different parts of M. alba (Cortex Mori, Ramulus Mori, Folium Mori and Fructus Mori) have different bioactivities; however, most current evidence is focused on anti-diabetic properties. In spite of their widespread use, few studies have compared the chemical composition and activity against α-glucosidase of the various medicinal parts of M.alba. In this study, we developed an HPLC method for simultaneous quality control and discrimination of Cortex Mori, Ramulus Mori, Folium Mori and Fructus Mori using 13 marker compounds. We found that quercetin, morin, kuwanon G, sanggenon C, morusin, mulberroside A and rutin were chemically distinct among the various medicinal parts of M. alba. A spectrum-effect relationship method was established to compare α-glucosidase inhibitory activity of various batches of samples to determine the activity of the primary active components against α-glucosidase. Taken together with molecular docking data, we found that prenylated flavonoids (morin, sanggenon C, kuwanon G and morusin), flavonols (kaempferol, quercetin, rutin and isoquercitrin) and alkaloids (1-deoxynojirimycin) were small molecule α-glucosidase inhibitory ingredients. In conclusion, we laid a solid foundation for effective substance identification in various parts of M. alba, and simultaneously provided a basis for their quality control.