Abstract: The paper reports the domestication,screening and combination of the toluene degrading bacteria and desulfurizing-bacteria,investigates the degradation conditions of mixed bacteria,with sodium thiosulfate and toluene as substrate,respectively. Four strains of efficient desulfurizing-bacteria(Z₁,Z₂, Z₃,and B₁) and three strains of toluene degradation bacteria(J₁,J₂,and J₃) were obtained by domesticating bacteria sources. Then,the two kinds of strains were combined to obtain the best combination mode for(Z₁+ Z₂)- B₁-(J₁+ J₃),and the density ratio of thallus 2:4:3. In addition, The central composite design(CCD) of response surface method(RSM) was used for optimizing the degradation condition of the mixed bacteria,SO₄ ^2-concentration and toluene degradation rate as control index. The optimum conditions were as follows:pH 6.8,temperature 30.0℃,inoculation amount 3.0%,toluene mass 3.0 μL/80 mL,and sodium thiosulfate mass 4.4 g/L. The verified experiment shows that toluene degradation rate can reach 73.9% and SO₄^2-concentration of 163.9 mg/L under the best degradation conditions,which was basically identical to the prediction results,and improved significantly compared with removal performance before optimization.