BACKGROUND: 2-Phenylethanol (PEA) is an important aromatic compound produced mainly by chemical synthesis. PEA production by biotechnological approach has gained popularity in recent years. However, biological synthesis of PEA is limited by microorganisms and product inhibition. The influence of culture temperature on the bioconversion of L-phenylalanine (L-Phe) into PEA is investigated in this study. A novel strategy to enhance PEA using two-stage fermentation with in situ product recovery also is proposed. RESULTS: Batch culture at 25 °C with aeration rate of 1.3 vvm was optimal for high cell density, whereas cultivation at 35 °C with aeration rate 2 vvm was favorable for high product yield. Then, a two-stage batch fermentation with switch time at 72 h was performed resulting in an increase in PEA concentration by 19.0% as compared to a single-stage fermentation at 35 °C with a 2.0 vvm aeration rate. A semicontinuous fermentation with in situ product recovery was conducted, yielding a total PEA concentration of 4.5 g L−1. CONCLUSIONS: The influence of culture temperature on bioconversion of L-Phe is significant. Cell growth favors a lower temperature at 25 °C, but product formation favors a higher temperature at 35 °C. A two-stage fermentation strategy was successful in batch culture, but not in fed-batch culture due to the product inhibition from the accumulation of PEA. Therefore, employment of two-stage semicontinuous culture with in situ product removal was successfully demonstrated in this study.