Background Natural 2-phenylethanol (2-PE) is an important flavoring that emits the aroma of roses. During biotransformation, the aroma quality of natural 2-PE is affected by its main by-products, which include butanol, isobutyric acid, butyric acid, and isovaleric acid. Thus, controlling undesirable by-product formation can reduce the effect of odor on 2-PE aroma quality. Results 2-PE was produced through biotransformation using l-phenylalanine as a substrate and glucose as a carbon source. Ascorbic acid was added to the system to improve the redox reaction and suppress the generation of by-products. Principal component analysis of the aroma quality of 2-PE was performed using an electronic nose. Similarity analysis revealed that the effects of four by-products on 2-PE aroma quality may be ranked in the following order: isovaleric acid > butyric acid > isobutyric acid > butanol. The sample that exhibited the best similarity to the standard 2-PE sample (99.19%) was the sample to which ascorbic acid had been added during glucose metabolism. Conclusions 2-PE produced through the addition of ascorbic acid exhibited the closest aroma similarity to the standard 2-PE sample.