The alpha-glucosidase AglA from Xanthomonas campestris suffers from low catalytic activity for alpha-arbutin production, which leads to unsatisfied productivity. To address this issue, herein, the critical amino acid residue Leucine145 of alpha-glucosidase AglA, which locates on the loop adjacent to maltose binding domain, is identified with potential capability of affecting the enzyme activity with the aid of computational analysis. Site-directed and saturation mutagenesis on this key residue is performed, and the best mutant L145V with 7.2-fold improvement in catalytic efficiency (kcat/Km) is achieved. Following maltose hydrolysis test and alpha-arbutin production by whole cell biotransformation further demonstrate that L145V has the most excellent catalytic performance. The production of alpha-arbutin produced by Escherichia coli BL21(DE3) cells harboring mutant L145V reaches 63.1 mM within 3 h, which is 4.7-fold higher than that of wild type AglA. Finally, molecular dynamics simulations studies provide molecular insights into the possible maltose binding poses, and shed light on the possible reasons behind the greatly enhanced activity of mutant L145V. These results reveal critical roles of residue L145 of alpha-glucosidase AglA in regulating catalytic activity, which will be very useful for guiding the further engineering of other glucosidases.