An online solid-phase microextraction coupled liquid chromatography-electrospray ionization-ion trap mass spectrometry was developed for the analysis of trace R- and S-4-phenyl-2-butanol (R- and S-pbol) in salt rich cell culture of Saccharomyces cerevisiae catalyzed stereoselective reduction of 4-pheny-2-butanone (pbone). A Supel-Q PLOT capillary column was used for the extraction and deionized distilled water was used as the extraction mobile phase. The extraction flow rate and extraction time were at 0.1mLmin−1 and 0.95min, respectively. The three target analytes, pbone, R-pbol, and S-4-pbol, were desorbed and eluted by the mobile phase of water/methanol/isopropanol (55/25/20, v/v/v) with a flow rate of 0.5mLmin−1 and analyzed by a chiral column. The mass spectrometric detection of the three target analytes was in positive ion mode with the signal [M+Na]+. The matrix-matched external standard calibration curves with linear concentration range between 0 and 50μgmL−1 were used for quantitative analysis. The linear regression correlation coefficients (r2) of the standard calibration curves were between 0.9950 and 0.9961. The yeast mediated reduction was performed with a recation culture of yeast incubation culture/glycerol (70/30, v/v) for 4 days. This biotransformation possessed 82.3% yield and 92.9% S-enantomeric excess. The limit of detection (LOD)/limit of quantification (LOQ) for pbone, R-pbol, and S-pbol was 0.02/0.067, 0.01/0.033, and 0.01/0.033μgmL−1, respectively. The intra-day and inter-day precisions from repeated measurements were 10.8–21.1% and 11.6–18.7%, respectively. The analysis accuracy from spike recovery was 84–91%.