Abstract: Fermentation broth of Streptomyces lydicus E12 displayed strong antagonistic activity against some pathogenic fungi. Effect of starch and glucose on the antifungal activity of fermentation broth was first explored by the agar diffusion method. Then antifungal substance was extracted from the fermentation broth by macroporous resin column chromatagrophy and gravity thickening. The molecular weight and β-1, 3-glucanase activity of the antifungal crude extract was detected through the reaction between the polyacrylamide gel and a thin agar gel containing β-1, 3-glucan. Results showed that the inhibition ratio of the fermentation broth dropped from 89.2% to zero when starch was replaced by glucose in the media as the solar carbon source. There was only one band on the SDS-PAGE gel with a molecular weight of about 3 kDa which showed antifungal activity, and at the corresponding position on the agar gel a clear band appeared. Take together, Streptomyces lydicus E12 can produce an antifungal substance with β-1, 3-glucanase activity.