Mulberry leaves have been used in the clinical treatment of diabetes since ancient times. A variety of bioactive components in mulberry leaf extract are effective α-glucosidase inhibitors. Though much research has been done on dry mulberry leaves, few have been done on the fresh mulberry leaves. In this study, Ganoderma lucidum was used to ferment fresh mulberry leaves, the effects of microbial fermentation on the content of active components and α-glucosidase inhibitory activity in dry and fresh mulberry leaves were measured. The results showed that, compared with dry mulberry leaves, fresh mulberry leaves were more suitable for the growth of Ganoderma lucidum. The nutrition needed for the growth of Ganoderma lucidum was mainly from the degradation of polysaccharides in fresh mulberry leaves. After fermentation, the polysaccharides content of fresh mulberry leaves decreased from 224.18 mg/g to 22.08 mg/g. The enrichment of bioactive components in fresh mulberry leaves was higher than that in dry mulberry leaves. The contents of flavonoids and alkaloids in fresh mulberry leaves were increased by 34% and 35% respectively compared with nonfermented mulberry leaves. The inhibitory effect of flavonoids and alkaloids on α-glucosidase of fresh mulberry leaves was significantly enhanced by fermentation. In conclusion, the fermentation of fresh mulberry leaves by Ganoderma lucidum fungus can not only enrich the content of α-glucosidase inhibitors in mulberry leaves, but also significantly improve their inhibitory activity.