The production of lactosucrose is hampered by the costly use of β-fructofuranosidase, which shows poor stability and a low efficiency in transfructosylation activity. Immobilization could improve enzyme stability and realize the cyclic utilization at a reduced cost. In order to eliminate the by-product inhibition and improve the transfructosylation efficiency, β-fructofuranosidase and glucose oxidase were co-immobilized by sol-gel encapsulation and the subsequent production efficiency of lactosucrose was investigated. The as-prepared immobilized bi-enzymes retained 85.39% of their initial activity at an enzyme concentration of 1.47 mg/g·sol during immobilization and showed great operational stability (maintaining 78.5% of their initial activity) after 15 consecutive reuses. The yield of lactosucrose synthesized by immobilized bi-enzymes reached 160.8 g/L under the optimized conditions, which was relatively higher than previous reported results. Moreover, the yield of lactosucrose synthesized by immobilized bi-enzymes was significantly improved as compared to that synthesized by immobilized β-fructofuranosidase. HPLC and NMR spectrum results confirmed the presence of lactosucrose during immobilized bi-enzymes catalysis. Furthermore, a relatively high purity of lactosucrose was obtained (87.4% determined by HPLC) after separation with Diaion UBK535 calcium ester resin, and the optimal conditions for separation of lactosucrose were investigated. These results indicated that the co-immobilization of β-fructofuranosidase-glucose oxidase was helpful to improve the production of lactosucrose with low costs, which can be used in continuous lactosucrose production in food industry in advantages of high stability and reusability. And the as-prepared lactosucrose with high purity can be applied to many kinds of food as functional additives.