(S)-Mandelic acid [(S)-MA] was prepared in a one-pot fermentation-transformation process by a (R)-MA-degrading bacterium, Pseudomonas putida ECU1009. Using a sequential simplex optimization method, the best results were obtained with growing cells cultivated on a medium composed of 1.0% (RS)-MA, 0.1% NH 4 NO 3 , and 0.1% corn steep liquor. The optimal cultivation conditions were 30°C and pH 6.5. By fed-batch culture under the optimal conditions, (S)-MA was successfully prepared in 1-L Erlenmeyer flask with 41% isolation yield and >99.9% ee, from totally 3% (RS)-MA after 94h of biotransformation.