Regioselective enzymatic glycosylation of D-glucose and various structural alcohols for synthesizing beta-D-glucopyranosides was successfully conducted. Among the beta-glycosidases from seven potential plant seeds, the low-cost beta-glycosidase from Prunus persic a seed displayed excellent activity for salidroside preparation. Several crucial parameters, such as the solvent, buffer pH, buffer content, substrate molar ratio, enzyme dosage, and temperature were examined for yield optimization. Under the optimal conditions, the initial reaction rate and yield of salidroside were as high as 3.04 mM/h and 18.90%, respectively. Moreover, the alcohol acceptor recognition of the enzyme in synthesizing beta-D-glucopyranosides was also investigated. The experimental data indicated that beta-glycosidase activity and yield varied widely among all the tested alcohols due to the specific spatial structures of various acceptors.