In this article, we report a method for preparing an immobilized bacterial agent of petroleum-degrading bacteriaGordonia alkanivoransW33 by combining high-density fermentation and bacterial immobilization technology and testing its bioremediation effect on petroleum-contaminated soil. After determining the optimal combination of MgCl2, CaCl2concentration, and culture time in the fermentation conditions by conducting a response surface analysis, the cell concentration reached 7.48 × 109CFU/mL by 5 L fed-batch fermentation. The W33-vermiculite-powder-immobilized bacterial agent mixed with sophorolipids and rhamnolipids in a weight ratio of 9:10 was used for the bioremediation of petroleum-contaminated soil. After 45 days of microbial degradation, 56.3% of the petroleum in the soil with 20,000 mg/kg petroleum content was degraded, and the average degradation rate reached 250.2 mg/kg/d.