The invention relates to transglucosidase, its preparation method and a immobilization method, which belong to the fields of enzyme engineering and fermentation engineering. More specifically, the invention provides the following processes: 1) Aspergillus niger BLB-16 is screened from soil as an original strain, processes of mutation treatment, optimization and screening are performed to obtain an optimized bacterial strain (BLB-28) for fermenting; 2) a fermentation broth is carried out a heat sterilization to obtain transglucosidase liquid; 3) transglucosidase liquid is performed a nanofiltration to concentrate; 4) processes of resin adsorption, sodium alginate entrapment, immobilization by a glutaraldehyde cross-linking method are carried out for preparing immobilized enzymes. The prepared transglucosidase is suitable for an application in the industrial fields such as foodstuff, medicine, feed and the like, and used for producing isomaltose hypgather.