Using of clearing zones on pachyman agar medium, there are 217 plant endophytic actinomycetes, producing β-1,3-glucanase were screened. 45.6% of the strains produced β-1,3-glucanase, in which the strains from cucumber are up to 38. The percentages of endophytic actinomycetes from different hosts produceing β-1,3-glucanases were different. The percentage of the strains in Rhizoma Polygonatum produced β-1,3-glucanases is the highest, up to 88.9%. The Inhibited effects of plant endophytic actinomycetes which produced extracellular β-1,3-glucanases on mycelium growth of Sclerotinia sclerotiorum were detected in vitro. Cucumber endophytic actinomycete gCLA4 strain was screened out from 99 isolates, which can strongly inhibit the growth of S. sclerotiorum. The optimal β-1,3-glucanases fermentation conditions of strain gCLA4 were investigated, they were pachyman 0.2%, peptone as nitrogen, pH 7~8 for 5 days. The β-1,3-glucanases of strain gCLA4 had some inhibiting efficiency on 13 plant pathogens, in which inhibiting efficiency to Botryosphaeria dothidea was the strongest.