Abstract: The Pseudomonas sp. LKY-5 produces biosurfactant during biodegradation process of dibenzothiophene. This work optimizes the carbon sources for biosurfactant production, verifies the biosurfactant by thin-layer chromatography, and then determines its physicochemical properties, and investigates the influence of temperature, pH and inorganic ions on the active stability of biosurfactant. The results show that the biosurfactant produced is rhamnolipid, and peanut oil is the optimal carbon source for the production of biosurfactant with a yield of 0.15 g/L. The critical micelle concentration (CMC) and hydrophile-lipophile balance (HLB) value of biosurfactant are 180 mg/L and 12.3, respectively. Twenty-four hours diesel emulsification ability is 61%. The biosurfactant is stable at conditions of 30~80 °C, pH of 6~13, and can tolerate the environment of 200 g/L NaCl (or MgCl2) or 20 g/L CaCl2.