background: 2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an important derivative of L-ascorbic acid (L-AA), which has the distinct advantages of non-reducibility, antioxidation, and reproducible decomposition into L-AA and glucose. Enzymatic synthesis is a preferred method for AA-2G production over alternative chemical synthesis owing to the regioselective glycosylation reaction. α-Glucosidase, an enzyme classed into O-glycoside hydrolases, might be used in glycosylation reactions to synthesize AA-2G. unassigned: Here, an α-glucosidase from Oryza sativa was heterologously produced in Pichia pastoris GS115 and used for biosynthesis of AA-2G with few intermediates and byproducts. The extracellular recombinant α-glucosidase (rAGL) reached 9.11 U mL after fed-batch cultivation for 102 h in a 5 L fermenter. The specific activity of purified rAGL is 49.83 U mg at 37°C and pH 4.0. The optimal temperature of rAGL was 65°C, and it was stable below 55°C. rAGL was active over the range of pH 3.0-7.0, with the maximal activity at pH 4.0. Under the condition of 37°C, pH 4.0, equimolar maltose and ascorbic acid sodium salt, 8.7 ± 0.4 g L  of AA-2G was synthesized by rAGL. conclusions: The production of rAGL in P. pastoris was proved to be beneficial in providing enough enzyme and promoting biocatalytic synthesis of AA-2G. These studies lay the basis for the industrial application of α-glucosidase.