Debranching enzymes are commonly used enzymes in the starch deep processing industry. However， traditional debranching enzymes are unable to fully meet the requirements of green production of starch sugars and comprehensive utilization of by-products. Therefore， it is necessary to explore suitable new types of debranching enzymes. This study screened an oligo-1，6-glucosidase gene (oga) from Paenibacillus sp. STB16 and constructed its Bacillus subtilis expression system. This system achieved heterologous expression of the oligo-1，6-glucosidase. The enzymatic properties and substrate specificity of the recombinant oligo-1，6-glucosidase were explored. The results showed that the enzyme activity of the fermentation supernatant of B. subtilis could reach 162.33 U/mL. The recombinant oligo-1，6-glucosidase had the optimum reaction temperature at 50 ℃ and optimum reaction pH at 6.0. It specifically acted on the α-1，6-glycosidic bonds of branches with degree of polymerization of 1. Compared to other types of debranching enzymes (substrate conversion rate under the detection limit)， the recombinant oligo-1，6-glucosidase exhibited stronger hydrolytic activity towards isomaltose， isomaltotriose， and panose. The substrate conversion rate reach 97.4%-100%， with glucose as the product.Therefore， it is more suitable for treating glucose mother liquor. The glucose content in the first mother liquor， second mother liquor and tail liquor of chromatographic separation was significantly increased by 3.6%， 12.7% and 34.4%， respectively. The research results provide a theoretical basis for the development and utilization of new types of debranching enzymes， and also serve as an important reference for the comprehensive utilization of by-products in starch sugar processing.