OBJECTIVE: To optimize the conditions of extractive microbial transformation for producing L-phenylacetylcarbinol (L-PAC). METHODS: HPLC was used to determine the concentration of L-PAC, which was extracted by saccharmyces cerevisiae. Using L-PAC concentration as response value, Box-Behnken response surface design was adopted to investigate the 3 main factors of benzaldehyde, Triton X-100 and glucose dose, and verification test was conducted. RESULTS: The interaction between benzaldehyde and Triton X-100 was the most significant. The optimized combination was as follows as benzaldehyde of 1.1%, Triton X-100 of 0.14 g/mL, glucose of 0.028 g/mL. The average concentration in verification test of L-PAC was 28.04 mmol/L (RSD=1.35%, n=3), showing 0.11% relative error with the predicted value of 28.01 mmol/L. CONCLUSIONS: Response surface method has optimized the conditions of extractive microbial transformation for producing L-PAC, obtained optimal combination of each factor, and provided favorable reference for volume-producing L-PAC.