<p>Crude petroleum pollution causes some serious ecological disasters in the ocean. Marine sediment microbial fuel cells (MSMFCs) have been utilized as a novel method for <i>in-situ</i> degradation and a long-term power source. Herein, the effect of different concentrations of rhamnolipids biosurfactant on the electrochemical performance of MSMFCs anode and the higher efficiency of oil degradation are creatively investigated. The results indicate that the anode in sediment containing rhamnolipids effectively enriches the indigenous electrogenic <i>Pseudophaeobacter</i> and <i>Pseudomonas</i>, which significantly enhances the electrochemical performance of the MSMFCs. Under rhamnolipids at the concentration of 200 mg kg⁻¹ in sediment, the anode specific capacitance (401.45 F m⁻²), exchange current density (4.87 × 10⁻² mA m⁻²), and cell maximum power density (140.24 mW m⁻²) increase by 2.50, 38.65, and 2.11 times, respectively, in comparison with its natural sediment. And the oil degradation rate (40.06%) was higher than the blank (17.55%). It demonstrates that the synergistic effect between electrochemical catalytic degradation and emulsifying solubilization of rhamnolipids surfactant directly accelerates the degradation of petroleum in marine sediment, which will provide a novel method and theoretical guidance for <i>in-situ</i> degradation and efficient removal of crude petroleum on ocean floor.</p>