Abstract: Rice bacterial leaf streak is an important plant disease caused by Xanthomonas oryzae pv. oryzicola, a pathogenic variant of X. oryzae. There is an urgent need to develop green, efficient, and low-toxicity biopesticides for its control. Bacillus altitudinis ST15 shows a significant inhibitory effect on X. oryzae and has the potential to develop into biopesticides. However, the low number of live bacteria and spores during shake-flask fermentation limited its development and application. Therefore, the effects of temperature, stirring speed, ventilation rate, and other parameters on the fermentation of ST15 were studied in a 5 L fermentator. The factors affecting the number of live bacteria and spores were identified. The results showed that the optimal temperature, agitation speed, ventilation flow, seed age, inoculation volume, and fermentation period were 30 ℃, 500 r/min, 4 L/min, 12 hours, 8%, and 36 hours, respectively. Under this condition, the number of viable bacteria and spores, as well as the spore-producing rate of ST15 reached 74.54 × 10⁸ CFU/mL, 64.75 × 10⁸ CFU/mL, and 86.91%, respectively. These values improved by 16.11%, 42.00%, and 27.75% compared with those under shake-flask fermentation conditions. These findings provide technical support for the subsequent pilot and large-scale production of ST15.