High cell density cultivation (HCDC) of desulfurizing cells of Rhodococcus erythropolis LSSE8-1 by Taguchi orthogonal array (OA) experimental design (DOE) methodology was investigated. Glycerol and ammonium chloride were found to be the optimal carbon and nitrogen sources for the growth of R. erythropolis LSSE8-1. Dimethylsulfoxide (DMSO) was more suitable than dibenzothiophene (DBT) as a sulfur source. Five factors viz. glycerol, ammonium chloride, DMSO, magnesium chloride, trance element solution at four levels with a OA layout of L16 (4⁵) were selected for the Taguchi DOE. The optimized culture conditions showed an enhanced cell production of 90.5% (from 2.61 to 4.98 g dry cells/L at 48h cultivation). High cell density cultivation of R. erythropolis LSSE8-1 using pH-stat and fed-batch feeding strategies was further validated in a 5 L fermentor with the optimal factors. As a result, a cell concentration of 23.86 g dry cells/L was obtained after 96h cultivation. Application of Taguchi approach appears to have potential usage in fermentation optimization. The resting cells and the straight fermentation suspension obtained from the HCDC were applied for the deep desulfurization of a hydrodesulfurized-treated diesel oil. Both of them showed high desulfurization activeity. It was observed that the sulfur content of the diesel decreased from 555.07 to 105.97 μg/g in 24h at a 1:2 oil-water ratio. It implied that the biodesulfurization process can be simplified by directly mixing cell cultivation suspension with diesel oil and the biocatalyst developed in this study had a good potential to be applied to a deep desulfurization process to product ultra-low-sulfur petroleum oils.