Abstract: In this work, biosurfactant-containing oxygen-releasing beads (BS-ORBs) were prepared and used for the bioremediation of BTEX in groundwater. The response surface methodology (RSM) was utilized to optimize the BS-ORBs. The results reveal that the optimal proportions of polyvinyl alcohol (PVA), biochar, CaO2, alginate, biosurfactant, and citrate were 33%, 32.5%, 13%, 13%, 5.8%, and 2.6% (w/w), respectively. The integrality ratio of optimized BS-ORB was 3.81%. The regression equation obtained from RSM accurately predicted the integrality ratio (R2 = 0.994). The BS-ORBs continuously released oxygen (>0.5 mg/L) for 128 days. The concentration of the biosurfactant was maintained above 30 mg/L for 58 days. Batch experiments indicated that adding BS-ORB improved the bioremedation of the groundwater that contained high levels of BTEX (120 mg/L) for three cycles. These results indicate that the BS-ORBs are a potential oxygen-releasing material for the biomediation of groundwater that is contaminated with a high concentration of BTEX.