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This study investigated the effect of immobilized biosurfactant-producing bacteria on the bioremediation of diesel oil-contaminated seawater. Initially, a biosurfactant-producing bacterium, LQ2, was isolated from a marine cold-seep region, and identified as Vibrio sp. The biosurfactant produced by LQ2 was characterized as a phospholipid, exhibiting high surface activity with strong stability. Meanwhile, the inoculation of biochar-immobilized LQ2 demonstrated superior efficiency in removing diesel oil (94.7%, reduction from 169.2 mg to 8.91 mg) over a seven-day period compared to free-cell culture (54.4%), through both biodegradation and adsorption. In addition, the microbial growth and activity were greatly enhanced with the addition of immobilized LQ2. Further experiment showed that degradation-related genes, alkB and CYP450–1, were 3.8 and 15.2 times higher in the immobilized LQ2 treatment, respectively, than those in the free cell treatment. The findings obtained in this study suggest the feasibility of applying immobilized biosurfactant-producing bacteria, namely LQ2, in treating diesel oil-contaminated seawater.