[Background] Natural production derived from microorganisms are one of the hot topics in the research and development of new pesticides. [Objective] To isolate and screen soil fungi producing potential herbicidal metabolites. [Methods] Petri dish filter paper culture method was adopted to determine the inhibition effect and stability of fermentation broth on the growth of plant seedlings, and the active strain was identified by microscopic observation and rDNA ITS sequence analysis. [Results] Of 30 fungal strains isolated from soil, the fermentation broth of isolated strain L-27 had significant inhibitory effect on wheat seedling, with inhibition rates 79.4% and 67.3% on root and stem length. Based on colony morphology, micromorphology and rDNA ITS gene sequences analysis, the isolated strain L-27 was identified as Aspergillus tubingensis. Further, strain L-27 fermentation broth completely inhibited seedling growth of Amaranthus retroflexus, Portulaca oleracea, Echinochloa crusgalli and Ipomoea purpurea except for 77.8% reduction of stem length on I. purpurea. The inhibitory effect of strain L-27 fermentation broth on wheat seedling growth had high stability to temperature, pH and UV light radiation, with inhibition rate 100% and 73.8% on root and stem length even heated at 120℃ for 20 min, 100% and 100% at pH 2.0—12.0 for 1 h, and 100% and 84.3%—91.7% under UV light radiation for 5 min to 240 min. [Conclusion] The fermentation broth characteristics of the isolated A. tubingensis L-27 indicated its great potential to develop herbicide.