Abstract: In order to control Aspergillus flavus growth and aflatoxin contamination more safely and effectively, a strain with excellent inhibitory effect on the growth of A. flavus was isolated from the soil of peanut field. The antagonistic effect against A. flavus was confirmed by plate confrontation examination; 16S rDNA sequence alignment analysis, physiological and biochemical characterization were employed for strain identification. Furthermore, the optimal medium for producing antibacterial substance was evaluated by spore germination investigation, and the inhibition effect of fermentation broth on mycelial growth and spore production of A. flavus was evaluated. In addition, the inhibitory mechanism was elucidated through PI and DAPI staining. The results showed that the colony growth of A. flavus was inhibited by strain 41 in a noncontact mode, indicating that strain 41 could antagonize A. flavus via producing some antifungal compounds. The physiological and biochemical analysis showed that strain 41 was a gram-positive bacterium and could utilize glucose, maltose, fructose, sucrose, and xylose, as well as hydrolyze starch. In salt-tolerance experiments, strain 41 grew normally in 2% NaCl, 5% NaCl, and 7% NaCl, but not in media containing 10% NaCl. Furthermore, strain 41 could inhibit spore germination of A. flavus in glucose medium, sucrose medium and beef extract medium, among which glucose medium was the most effective, and the inhibitory effects of the fermentation broth on the mycelial growth and spore production of A. flavus were investigated under this condition. The inhibition rate of A. flavus spore germination was 79.35% by fermentation broth obtained in glucose medium of the isolated strain for 48 hours. On solid PDA medium, the inhibition rate of A. flavus colony growth and spore production was 29% and 54% respectively. The results of PI and DAPI staining on the mycelia treated with the fermentation broth showed that the antibacterial substances in the fermentation broth could inhibit the growth of A. flavus mycelia by destroying the permeability of cell membrane and damaging DNA respectively. This study demonstrated that the strain and its fermentation broth had an excellent inhibitory effect on A. flavus and provided a theoretical basis for development of the antifungal substance as a mildew inhibitor in agricultural field.