In order to achieve high expression of Trichoderma viride β-glucosidase in recombinant Pichia pastoris,the fermentation conditions of the P. pastoris KM71/pPIC9K-bgl1/pPICZ A-pdi were optimized in a 3.6L bioreactor. When the optimal conditions were the induction temperature 28℃, initial cell density 50g/L, and methanol volume fraction 1.0%, fermentation of P. pastoris KM71/ pPIC9K-bgl1/pPICZ A-pdi yielded an enzyme activity of 1452U/mL. β-glucosidase from the recombinant strain was immobilized on chitosan cross-linked with glutaraldehyde. When the optimal conditions of immobilization were 0.03g/mL chitosan, 0.008g/mL glutaraldehyde, 400U/g chitosan beads, and adsorption time 20h, the immobilization efficiency reached up to 65.4%. The yield ratio was still up to 15.2% after 6 times of continuous conversion and it showed a good operational stability and a high productivity of gentiooligosaccharides.