The immobilization of Pseudomonas delafieldii R-8 in calcium alginate beads has been studied in order to improve biodesulfurization activity in oil/water (O/W) biphasic systems. A gas jet extrusion technique was performed to produce immobilized beads. The specific desulfurization rate of 1.5 mm diameter beads was 1.4-fold higher than that of 4.0 mm. Some nonionic surfactants can significantly increase the activity of immobilized cells. The desulfurization rate with the addition of 0.5% Span 80 increased 1.8-fold compared with that of the untreated beads. The rate of biodesulfurization was markedly enhanced by decreasing the size of alginate beads and adding the surfactant Span 80, most likely resulting from the increasing mass transfer of substrate to gel matrix.