2-O-(α-d-glucopyranosyl)-sn-glycerol (2-αGG) has been applied in the food industry due to its numerous physiological benefits. The synthesis of 2-αGG can be achieved through a cascade catalytic reaction involving sucrose phosphorylase (SP) and 2-O-α-glucosylglycerol phosphorylase (GGP). However, the low substrate transfer rates between free enzymes have hindered the efficiency of 2-αGG synthesis. To address this issue, a novel technology was developed to prepare sequential multi-enzyme nanoflowers via chemical crosslinking and protein assembly, thus overcoming diffusion limitations. Specifically, spatially sequential co-immobilized enzymes, referred to as SP-GGP@Cap, were created through the targeted assembly of Bifidobacterium adolescentis SP and Marinobacter adhaerens GGP on Ca 2+ . This assembly was facilitated by the spontaneous protein reaction between SpyTag and SpyCatcher. Compared to free SP-GGP, SP-GGP@Cap demonstrated improved thermal and pH stability. Moreover, SP-GGP@Cap enhanced the biosynthesis of 2-αGG, achieving a relative concentration of 98 %. Additionally, it retained the ability to catalyze the substrate to yield 61 % relative concentration of 2-αGG even after ten cycles of recycling. This study presents a strategy for the spatially sequential co-immobilization of multiple enzymes in a confined environment and provides an exceptional biocatalyst for the potential industrial production of 2-αGG.