[Objective] The response surface methodology was used to optimize thermotolerant Pseudoxanthomonas oxidation effect. [Methods] We use Plackett-Burman method to select key factors for the sulfur oxidation. The steepest ascent method has been used to determine the optimal range of values and confirm the central axis. Box-Behnken experimental design was used to confirm the optimal concentration of the key factors. The concentration of sulfur was determined by classic barium sulfate turbidity method. [Results] Beef extract, malt sugar and Mg2+ were the key factors affecting the sulfur oxidation performance. The results of the response surface methodology showed that the interaction effect between beef extract and Mg2+ had the most effects on sulfate conversion rate. The optimization results were: malt sugar (%)=0.07, beef extract (%)=0.11, Mg2+=0.04, the sulfate conversion rate reached peak value. The F-value of the model was 52.60 (P<0.000 1) and correlation coefficient (R2)=0.980 2 which indicated the model terms were significant and showed good degree of fitting. The simulation composting experiments results showed that the concentration of sulfate in compost increased with Pseudoxanthomonas addition. [Conclusion] This model could be used to analyze and calculate the optimal formula of thermotolerant Pseudoxanthomonas culture medium. The sulfur conversion rate increased from 36.89% to over 80% after optimization. Addition of Pseudoxanthomonas in compost could increase sulfate concentration, compared with the suspension culture under basic fermentation conditions, which was of good application prospects.