Abstract: 【Objective】 To screen Trichoderma strains that could efficiently antagonize Alternaria alternata for biological control， the causa-tive agent of tobacco brown spot disease， and to investigate the enzymatic activity， antifungal acti-vity， and metabolite production of the selected Trichoderma strains， thereby providing theoretical basis for the biological control of tobacco brown spot disease and other diseases. 【Method】 Trichoderma strains were isolated from the rhizosphere soil of tobacco by dilution plate method. Antagonistic Trichoderma strains were screened by plate confrontation culture methods. The taxonomic status of the antagonistic Trichoderma strains was identified by morphology and multi-gene phylogenetic analysis. The cellulase， laccase， amylase， and siderophore production abilities of the antagonistic Trichoderma strain were determined by qualitative and quantitative analysis methods. Their antagonistic activity and stability against Alternaria alternata were evaluated using the mycelial growth rate method. Lastly， the metabolite components in the fermentation products of the Trichoderma strains were detected by LC-MS/MS technology. 【Result】 A total of 28 Trichoderma strains were isolated from the rhizosphere soil of tobacco， and a strain GF-13 with antagonistic effect against Alternaria alternata was screened. The confrontation culture test showed that the inhibition rate of strain GF-13 against the pathogen was 88.48%， and the dual culture test showed that the inhibition rate was 34.17%. Through morphological and multi-gene phylogenetic analysis， strain GF-13 was identified as Trichoderma asperellum. The enzymatic activity test results showed that the endoglucanase， exoglucanase， and filter paper enzyme activities of strain GF-13 were 0.76， 0.63， and 0.51 U/mL respectively， the laccase activity was 99.30 U/mL， and the siderophore content （on the 8^th d） was 76.69%， and it also had amylase activity. Mycelial growth rate method tests indicated that the inhibition rates of the crude extract of the fermentation broth and the mycelial crude extract of strain GF-13 against Alternaria alternata were 69.33% and 53.67% respectively. The crude extract of the fermentation broth displayed good tolerance to temperature， acidity， and alkalinity variations but showed poor tolerance to ultraviolet radiation. LC-MS/MS detection and analysis results confirmed that the fermentation broth and mycelial crude extract of strain GF-13 contained antibacterial and growth-promoting active substances， such as 2-hydroxycinnamic acid， betaine， gibberellin A43， and zeatin. 【Conclusion】 Trichoderma asperellum GF-13 has the ability to synthesize various antibacterial and growth-promoting active substances， and has a good application prospect in crop production and disease control.