β-Glucosylglycerol (β-GG) and their derivatives have potential applications in food, cosmetics and the healthcare industry, including antitumor medications. In this study, β-GG and its unnatural glycosides were synthesized through the transglycosylation of two enzymes, Sulfolobus shibatae β-glycosidase (SSG) and Deinococcus geothermalis amylosucrase (DGAS). SSG catalyzed a transglycosylation reaction with glycerol as an acceptor and cellobiose as a donor to produce 56% of β-GGs [β-D-glucopyranosyl-(1→1/3)-D-glycerol and β-D-glucopyranosyl- (1→2)-D-glycerol]. In the second transglycosylation reaction, β-D-glucopyranosyl-(1 → 1/3)-Dglycerol was used as acceptor molecules of the DGAS reaction. As a result, 61% of α-Dglucopyranosyl-( 1→4)-β-D-glucopyranosyl-(1→1/3)-D-glycerol and 28% of α-D-maltopyranosyl- (1→4)-β-D-glucopyranosyl-(1→1/3)-D-glycerol were synthesized as unnatural glucosylglycerols. In conclusion, the combined enzymatic synthesis of the unnatural glycosides of β-GG was established. The synthesis of these unnatural glycosides may provide an opportunity to discover new applications in the biotechnological industry.