Dibenzothiophene and its derivatives are components of high-sulfur crude oil, and these sulfides would reduce the oil quality and increase sulfur dioxide emissions when burned, which will decline environmental quality. Compared with the traditional desulfurization method, biodesulfurization has many advantages, but how to improve the biological desulfurization efficiency has been an important issue. In this study, culture conditions and desulfurization conditions were optimized by response surface methodology (RSM) to improve the biological desulfurization efficiency. Based on the single-factor experiments, Plackeet-Burman method (PB) was used to determine the main factors which would influence the efficiency of desulfurization. And the three main factors were temperature, pH and DBT concentration. Response surface optimization experiments show that when temperature was 29.70°C, initial pH was 7.43, and DBT concentration was 105.47mg/L, the desulfurization rate would be maximum, theoretical maximum degradation rate were 72.32%, and actual degradation rate were 74%. The interaction between the three key factors was significant, the influence degree was DBT concentration> pH> temperature. This study provides a method to improve the biological desulfurization efficiency by optimizing culture conditions and desulfurization conditions.