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In the recent 50 years, marine oil spills had resulted in severe environmental pollution problems worldwide. In this study, 12 petroleum-degrading strains named MJ1 to MJ12, which can use diesel oil as the sole carbon source for growth, were isolated from the seawater in Jiaozhou Bay, China. Strain MJ4 has the highest diesel-degrading rate which is up to 26.54% in 5 days with the diesel oil concentration of 10 g/L. According to the BLAST research, 16SrRNA sequence of MJ4 showed 99% similarity to Bacillus megaterium strain. Single-factor experiments and response surface methodology were carried out to optimize the environmental factors and their reciprocal action for affecting the diesel oil degradation process of Bacillus sp. MJ4. Results of single-factor experiments revealed that the highest degradation rate was obtained with temperature of 28 °C, pH of 8.8, diesel oil concentration of 25 g/L, P/N ratio of 0.56, nitrogen and phosphorus dosage quantity of 0.35 g/L and 0.18 g/L, respectively. A nonlinear regression equation of diesel oil degradation rate and pH, temperature, P/N ratio was obtained. The model predicted the maximum degradation rate of 72.21% with temperature of 28 °C, pH of 8.88, P/N ratio of 0.31, respectively.