Microbial ecological agents are environmental friendly alternatives to antibiotics, which could be utilized as feed additives for preventing disease and promoting growth of animals. Bacillus subtilis could generate spores, which have strong resistance to environmental stress including heat in drying process, mechanical force in granulation process and acid in digestive tract. Therefore, Bacillus subtilis is one of the preferred strains for microbial ecological agents preparation. Sporulation rate and viable cell number determine quality of Bacillus subtilis microbial ecological agents significantly. Increasing sporulation rate is considered as one of the key methods to improve the quality of Bacillus subtilis microbial ecological agents. Molecular mechanism of sporulation process was presented and the factors regulating sporulation were discussed. Different fermentation methods for microbial ecological agents production were compared. Furthermore, a systematic description was given of the process optimization for enhancing effective cell number of Bacillus subtilis. At last, applications of Bacillus subtilis microbial ecological agents were presented and the future research directions were proposed.