BACKGROUND: Deep desulfurization of hydrocarbon fuels is receiving increasing worldwide attention because of the increasingly stringent regulations to meet the requirement of environmental protection. Biodesulfurization (BDS) is being explored as either an alternative or complementary process to the conventional oil refining technologies. The whole cell immobilization technique is of great importance for accelerating the industrialization of BDS. An effective technique for a BDS process employing flocculation and integration with immobilization was developed. RESULTS: Pseudomonas delafieldii R-8 cells were successfully flocculated and immobilized by directly adding chitosan and celite into the culture broth. The one-step immobilized R-8 cells exhibited good catalytic activity and retained at least 85% activity after six cycles of repeated-batch desulfurization. Extensive biodesulfurization of diesel oil resulted in 82% reduction of total sulfur from 123 to 22 ยตg g -1 in 24 h. CONCLUSIONS: A novel and simple technique was developed using chitosan flocculation and integration with cell immobilization onto celite for dibenzothiophene BDS. The present report indicates that integration of flocculation and immobilization may provide a continuous and efficient method of BDS.