Biodelfurization of petroleum has emerged as a potential alternative to the hydrodesulfurization and oxidative desulfurization processes. However, the main obstacle in its commercial application is the efficiency and practicality of using bacterial cells. Pseudomonas sp. strain KWN5 was tested for the ability to use dibenzothiophene (DBT) in n-tetradecane as the sole sulfur source with two phase oil-water system. The biodesulfurization ability of strain KWN5 was evaluated by immobilized cells with dibenzothiophene as substrates. The cells immobilized by entrapping them with sodium alginate (SA) had high DBT biodesulfurization activity and could degrade 100 mg DBT/L in n-tetradecane of 46.76–100%, depended on concentrations of sodium alginate and cells within 24 h at 37oC with shaking at 160 rpm. The combination of SA concentration of 3% (w/v) with bacterial cells OD660 40 (25.52 mg DCW/mL) has an optimal biodesulfurization activity on 100 mg DBT/L in n-tetradecane, which is equal to 71.85% biodesulfurization. The immobilized cells of Pseudomonas sp. strain KWN5 in alginate beads were more efficient for the degradation of DBT and can be reused for five cycles (220 h) without any loss in their activity. The results of this study clearly show the role of the effects of cell immobilization in increasing the process of biodesulfurization.