β-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg) and MEIR (9003.1 U mg) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105,836 U mL). The prebiotic mannooligosaccharides (<2000 Da) were prepared by hydrolyzing konjac gum and locust bean gum with MEIR, with 100% and 76.40% hydrolysis rates, respectively. These characteristics make MEIR highly attractive for prebiotic development in food and related industries.