This research aimed to optimize the experimental conditions of biodesulfurization of sulfide ore and to evaluate the flame-retardant effect after desulfurization under optimal conditions. Six experimental factors were determined: particle size of ore sample, ambient temperature, rotary speed of the shaking table, volume of bacteria liquid, concentration of leaching aid (Tween80), and pH value of acidizing ore sample. Desulfurization efficiency was used as the main characterization index of the desulfurization effect in optimization studies. Particle size of ore sample, rotary speed of the shaking table, and volume of bacteria liquid inoculated were selected as significant factors by a Plackett–Burman experiment. Modeling, optimization, and analysis of the interactive effects of these factors, notably between particle size and bacteria liquid, were performed using a Box–Behnken design with response surface methodology. The optimum operating conditions were found to be: particle size of 120 to 140 mesh, rotary speed of 175 rpm, and bacteria liquid of 111 mL. Under these conditions, a significant rise of 8.1% was seen in 5-day average desulfurization efficiency. The 5-day oxidation weight gain rate of desulfurized ore was 2.73%, while that of the control group was 4.78%. The results show that, after optimized desulfurization, the surface oxidizability and spontaneous combustion tendency of the ore are reduced.