Enantiopure esomeprazole is an important drug in the treatment of gastric ulcer. The asymmetric sulfoxidation of omeprazole thioether was catalyzed by immobilized cells of a mutant of Rhodococcus rhodocrous ATCC 4276 to synthesize esomeprazole. The bioreaction was carried out in a biphasic system (chloroform-water), at a high substrate concentration (200mM), and optimized using response surface methodology (RSM). The optimal yield of esomeprazole obtained was 94.8% with e.e. (>99%) without the formation of the sulfone form as a byproduct, under the optimal conditions: the concentration of immobilized cells, 283.5 g/L, the incubation temperature, 37.05 °C, and pH of phosphate buffer, 7.35, respectively. A quadratic polynomial model was developed with R2 of 0.9998, which indicates that the model predicts the observed data with very high accuracy. The mutant exhibited a high enantioselective activity and substrate and product tolerance. The small size of immobilized cell beads (0.5-1 mm) creates a large reaction interface. The aerated flask provides enough oxygen for a high concentration of cells. The significant improvement of substrate tolerance may mainly be attributed to employing the chloroform-water biphasic system because organic substrates may be partitioned in the organic phase, eliminating potential damage and inhibition to cells. Based on the above, the asymmetric sulfoxidation catalyzed by immobilized bacterial cells is therefore more promising for efficient synthesis of chiral sulfoxides.