Many studies have been conducted on the removal of heavy metals from industrial wastewater using biosurfactants, but few papers have been published on the removal of heavy metals from soil contaminated with crude oil using this method. The aim of this study is to evaluate the performance of rhamnolipid biosurfactants in removing nickel, chromium, and cadmium from soil contaminated with crude oil. In this study, first rhamnolipid was produced from Pseudomonas aeruginosa PTCC1340 and then confirmed by Thin-Layer-Chromatography (TLC) and Fourier-Transformation-Infrared-Spectroscopy (FTIR). In the next step, a sandy soil (2 mm diameter) was mixed with the crude oil (API = 32.83, viscosity = 6.21cp) at 258C for 2 weeks. The washing parameters included temperature, pH and rhamnolipid dosage. Based on our results, the heavy metal removal of nickel, chromium and cadmium from soil contaminated with crude oil at the optimum condition (258C, 0.8 g/L and pH 11) was 43.05%, 34.73% and 52.81%, respectively. Based on the results, rhamnolipids are found to have considerable potential for removing heavy metals especially cadmium, and the soil washing by the produced biosurfactant leads to the removal of heavy metals without the destructive effects of chemical surfactants and subsequently reduces environmental pollution. Therefore, this biosurfactant rhamnolipid may have a good potential for application in bioremediation of crude oil contamination.