Rebaudioside A with highly purity from commercialized stevia extracts is not amenable to preparative separation, owing to the structural similarity between stevioside and rebaudioside A, necessitating a new bioconversion method for its easy and simple separation. Complex stevia extracts were separated soluble or insoluble fractions after bioconversion with Penicillium decumbens‐derived β‐glucosidase (SPGase). Then, rebaudioside A or steviol was main component in the water soluble or insoluble. At 100 g/L commercialized stevia extracts scale, the enzymatic bioconversion gave the rebaudioside A (6.95 g/L) from soluble with highly purity and steviol (20.45 g/L) from insoluble.

This one step‐enzymatic bioconversion with SPGase could be used for the separation of rebaudioside A from commercialized stevia extracts. This simple method is very useful application for low‐calorie sweetener in food materials.