A lab-scale membrane-aerated biofilm reactor (MABR), inoculated with ADB350 M (the engineering bacteria on degrading petroleum hydrocarbon), was developed to process the oil-spilled seawater pretreated by rhamnolipid. Effects of the rhamnolipid/oil ratios, nitrogen and phosphorus additions, aeration pressures, and setup rotation velocities on the MABR performance were investigated. Results showed that under the optimal conditions, effluent concentrations of chemical oxygen demand (COD), oil content, total phosphorus (TP), and total nitrogen (TN) were 39 mg/L, 2.3 mg/L, 0.29 mg/L, and 1.9 mg/L, respectively. Throughout the treatment, the ADB350 M exerted good adherence to the aerated membranes. In comparison with the direct-spread of engineering bacteria, the membrane-immobilization method reduced losses of the inoculums inside the seawater. Supplement of rhamnolipid was conducive to the improvement of the biofflm hydrophobicity by changing the polysaccharide/protein ratios of extracelluar polymeric substances (EPS). In conclusion, the MABR system substantially removed oil content and other pollutants during the study, and it is of great value to explore the scale-up of this innovative technique for the oil-contained seawater bioremediation.