The invention relates to cotton verticillium wilt antagonistic bacillus subtilis, a fermented filtrate, and applications of the cotton verticillium wilt antagonistic bacillus subtilis and the fermented filtrate, the cotton verticillium wilt antagonistic bacillus subtilis is assigned with the accession number of CGMCC No.12885, and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on Aug 22, 2016. The cotton verticillium wilt antagonistic bacillus subtilis is separated from the cotton rhizosphere soil. The cotton verticillium wilt antagonistic bacillus subtilis has the obvious antagonistic effect for pathogenic bacteria of cotton verticillium wilt, has the inhibiting ratio for the mycelial growth of the cotton verticillium wilt reaching 51.23%-56.25%, and can promote seedling emergence of cotton, the screened cotton verticillium wilt antagonistic bacillus subtilis is separated from the cotton rhizosphere soil, and is safe to the plant, environment and human beings, resistance to drugs can not be caused to human beings, no hypertoxicity exists, and no hazardous substance is generated in the use process. The inhibition ratio of the fermented filtrate of the cotton verticillium wilt antagonistic bacillus subtilis for the spore germination of the pathogenic bacteria of cotton verticillium wilt is 78.54%-92.47%, and the fermented filtrate also can promote seedling emergence of cotton.