Bitumen contains approximately 5 wt% sulfur so it must be removed before being used as fuel. Herein, we performed oxidative desulfurization of bitumen at room temperature using n-pentane and ultrasound to reduce bitumen viscosity and mix the bitumen-dissolved n-pentane and aqueous reaction solution physically. The oxidative states of sulfur in bitumen and desulfurization ratio were investigated to confirm the oxidative desulfurization process. Additionally, we developed a novel method for separating bitumen into maltene and asphaltene. Thus, 81.0% of sulfur in maltene was successfully removed by removing asphaltene, which is difficult to remove sulfur using n-pentane in oxidative desulfurization.