Objective The existing position and extraction method of antifungal substance produced by Chaetomium subaffine strain LB-1 were studied in order to lay a foundation for the development of the strain LB-1 to control plant diseases.Method With Botrytis cinerea and Exserohilum turcicum as test plant pathogens, the sealed plate assay was used to detect whether the strain LB-1 could produce volatile antifungal substance. The hyphae ultrasonic breaking and liquid culture methods were used to detect the existing position of nonvolatile antifungal substance produced by the strain LB-1. The extraction method of antifungal substance was determined by detecting the inhibitory effects of ammonium sulfate precipitation, hydrochloric acid precipitation and organic solvents extracts of strain LB-1 culture broth via poison plate assay and filter paper disc assay.Result The strain LB-1 did not have obvious inhibitory effect on the growth of the two test plant pathogens when being co-cultured in a sealed plate with each plant pathogen, indicating that strain LB-1 could not produce volatile antifungal substance. The antifungal activity of the intracellular extract of the strain LB-1 was not different from that of the control, but its culture broth had a strong inhibitory effect on B. cinerea and E. turcicum, indicating that the antifungal substances produced by the strain LB-1 existed outside the mycelium. Neither ammonium sulfate precipitate nor hydrochloric acid precipitate of strain LB-1 culture broth showed inhibitory effect on B. cinerea and E. turcicum, but the organic solvent extract of strain LB-1 culture broth showed antifungal effect, and the inhibition rate of n-butanol extract was the highest. When the concentration was 0.1 mg/mL, the inhibition rates against B. cinerea and E. turcicum growth were 59.80% and 58.37% respectively.Conclusion The strain LB-1 inhibited the growth of plant pathogenic fungi by producing extracellular nonvolatile antifungal substances, and the antifungal substance in the culture broth can be extracted by n-butanol.