Biodesulfurization (BDS) is a promising technology for deep desulfurization. In this work, Pseudomonas delafieldii R-8 cells are immobilized in calcium alginate beads and used for BDS of transportation fuels. It is found that thiophene and dibenzothiophene (DBT) can be simultaneously metabolized by immobilized R-8 cells. The initial sulfur content in the model oil is 300 mg·kg-1 (thiophene " DI3T= 1 " 1). After 10 h of treatment, the thiophene concentration is reduced by 40%, while DBT is reduced by 25%. The utilization rate of thiophene is faster than that of DBT. Moreover, the oil/water ratio of alginate immobilized cells is studied to reduce the water volume in desulfurization systems. Long-term recycling of BDS by alginate immobilized cells is carried out with oil/water ratio at 5 : 1. The immobilized cells are successfully reused over 15 batch cycles. In the last batch, the desulfurization activity remains at least 75% of the first batch.