Abstract: To address the potential pollution issues caused by high-sulfur and -aluminum coal gangue stockpiles and explore their potential for kaolin extraction, this study developed a novel microbial method to treat coal gangue for synergistic desulfurization and iron removal. Using Acidithiobacillus ferrooxidans (WH1 strain, CGMCC No. 36180) isolated and cultured in situ from a coal gangue in a certain place in Inner Mongolia, single-factor experiments were firstly conducted to investigate the effects of initial pH, temperature and time on WH1's oxidative activity and sulfur-iron removal efficiency from coal gangue. Process conditions were then optimized using the Box-Behnken response surface method, combined with XRD, SEM-EDS, and ICP-OES to analyze synergistic deep removal behavior and mineral phase evolution characteristics. Results indicate that WH1 exhibits a logarithmic growth phase of 20-60 h in 9K medium, with peak oxidation activity at initial pH 2 and 30 °C, achieving the complete oxidation of ferrous iron within 96 h. Under the same conditions in a coal gangue nutrient-poor system, WH1 still exhibits excellent oxidation performance of ferrous iron, with sulfur and iron removal rates of 94.45% and 76.84% respectively after 21 d. XRD analysis results reveal the disappearance of characteristic pyrite peaks. Response surface regression models effectively fit, predict and guide the reaction parameter optimization. Under validated conditions of initial pH 1.96, 28.86 °C, and 18 d, sulfur and iron removal rates reach 97.2% and 88.7%, respectively, with deviations <1% from model predictions (sulfur 97.71%, iron 89.24%). The sulfur and iron removal behaviors exhibits strong positive correlation, confirming that WH1 bacteria removes sulfur and iron simultaneously primarily through pyrite oxidation. However, the presence of insoluble secondary jarosite minerals formed during microbial acid leaching and the original hematite in the coal gangue acts as limiting factors for deep removal of sulfur and iron from coal gangue. Kaolin prepared by calcining coal gangue under optimal conditions achieves a whiteness of 82%, and the other relevant technical indicators all meet the requirements of the “Calcined Kaolin Powder for Rubber and Plastic Industries” standard. This provides an economically feasible technological pathway for the resource utilization of high-sulfur and -aluminium coal gangue.