In this study, an acidophilic GH5 β-mannanase (TaMan5) fromTrichoderma asperellumND-1 was efficiently expressed inPichia pastoris(a 2.0-fold increase, 67.5 ± 1.95 U/mL). TaMan5 displayed the highest specificity toward locust bean gum (Km= 1.34 mg/mL,Vmax= 749.14 μmol/min/mg) at pH 4.0 and 65°C. Furthermore, TaMan5 displayed remarkable tolerance to acidic environments, retaining over 80% of its original activity at pH 3.0–5.0. The activity of TaMan5 was remarkably decreased by Cu2+, Mn2+, and SDS, while Fe2+/Fe3+improved the enzyme activity. A thin-layer chromatography (TLC) analysis of the action model showed that TaMan5 could rapidly degrade mannan/MOS into mannobiose without mannose via hydrolysis action as well as transglycosylation. Site-directed mutagenesis results suggested that Glu205, Glu313, and Asp357of TaMan5 are crucial catalytic residues, with Asp152playing an auxiliary function. Additionally, TaMan5 and commercial α-galactosidase displayed a remarkable synergistic effect on the degradation of galactomannans. This study provided a novel β-mannanase with ideal characteristics and can be considered a potential candidate for the production of bioactive polysaccharide mannobiose.