Gentiobiose is the main component of gentio-oligosaccharides, and it has excellent prebiotic functions. To improve the biosynthetic efficiency of gentiobiose and reduce production costs, the β-glucosidase gene was cloned from Aspergillus niger, and its recombinant expression was achieved using the Pichia pastoris expression system. The recombinant enzyme was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and its enzymatic properties were characterized. Using glucose as the substrate, key reaction parameters, including substrate concentration, temperature, pH, and enzyme dosage, were first evaluated by single-factor experiments, followed by optimization using an orthogonal experimental design. The results indicated that the specific activity of the purified recombinant β-glucosidase was 197.59 U•mg-1, and the optimal temperature and optimal pH were 60 ℃ and 4.5, respectively. The optimized conditions for gentiobiose synthesis demonstrated that, at an enzyme dosage of 105 U•g-1, a substrate concentration of 850 g•L-1, a temperature of 60 ℃, and a pH of 5.0 for 48 h, the yield of gentiobiose reached 163.40 g•L-1 and the conversion rate was 19.22%. In conclusion, the recombinantly expressed β-glucosidase exhibited high enzymatic activity and efficiently catalyzed the synthesis of gentiobiose, thereby providing a theoretical foundation for its biological preparation.