The present study shows the purification of a main oligosaccharide fraction (MLO 1-2) from the enzymatic hydrolysate of mulberry leaf polysaccharides by DEAE-52 cellulose and gel column chromatography. The physicochemical properties of MLO 1-2 were characterized. The structure of MLO 1-2 was obtained as follows: α-(2-OAc)-Manp-1 → 2-β-Glcp-1 → 4-β-Glcp-1 → 4-α-Glcp-1 → 2-α-Glcp-1 → 2-α-Galp-1 → 2-β-Galp-1 → 2-β-Galp-1, which was elucidated by methylation and NMR analysis. The molecular weight of MLO 1-2 showed no significant change after simulated saliva, gastric and intestinal digestion. This indicated that MLO 1-2 could pass through the digestive system without being degraded to safely reach the colon to regulate the gut microbiota. Additionally, MLO 1-2, more than glucose or galactooligosaccharides, promoted the proliferation of Bifidobacterium bifidum, B. adolescentis, Lacticaseibacillus rhamnosus and Lactobacillus acidophilus. Furthermore, the acetic and lactic acid concentrations of bacterial cultures inoculated with MLO 1-2 were higher than those inoculated with glucose and galactooligosaccharide (GOS). These results suggest that MLO 1-2 could be an excellent prebiotic for intestinal flora regulation and the promotion of gut health.