Abstract: 【Objective】This study aimed to clarify the classification status and biological characteristics of the pothogen of walnut leaf spot disease in Guizhou and screen effective fungicides，so as to provide theoretical basis for controlling the walnut leaf spot disease in the field.【Method】The leaves of walnut leaf spot disease collected from walnut planting base in Hezhang，Bijie，Guizhou were used as materials. Tissue separation method was used to isolate and purify the pathogenic bacteria，and Koch’s rule was used to determine the pathogenicity of the pathogen. The sequences of ITS， β-tubulin and TEF1-α genes in the pothogen were amplified respectively. Based on morphological characteristics and multigenes sequence analysis，the pathogen was classified and identified. Biological characteristics of the pathogen was determined by one-way variation method. Indoor toxicology test was conducted to evaluate the inhibtion effects of 17 common fungicides on leaf spot disease.【Result】Eighteen fungal isolates were obtained from the diseased walnut leaves in Hezhang，Bijie，Guizhou，and representative strain C5 was selected for morphological and pathogenicity determination. The pathogenicity detection results indicated that strain C5 was the pathogen causing walnut leaf spot disease. Morphological characteristics of strain C5 was similar to those of Fursarium equiseti，and multigenes cluster analysis showed that the pathogen was clustered in the same branch with F. equiseti yl-1. The optimum growth temperature of strain C5 was 20- 25 ℃，the optimum photoperiod were 18 h∶6 h（light∶dark）. Diverse carbon and nitrogen resources could be used，the optimum carbon source was sucrose，the best nitrogen source was beef paste. The germination rate of conidia was the highest under 70% humidity. Among the tested 17 fungicides，45% prochloraz ME，30% pyraclostrobin·tebuconazole SC，0.15% tetramycin AS，40% myclobutanil SC，10% difenoconazole WG and 1% phenazino-1-carboxylic acid SC had better inhibitory effects on the growth of strain C5 mycelium，and their median inhibitory concentrations（EC₅₀）were 0.0006，0.0016，0.0046，0.0062，0.0084 and 0.0097 μg/mL respectively.【Conclusion】The pathogen of leaf spot disease from the walnut growing area in Hezhang，Bijie，Guizhou is F. equiseti. 45% prochloraz ME or 0.15% tetramycin AS can be the first choice for controlling the disease，and 30% pyraclostrobin·tebuconazole SC，40% myclobutanil SC，10% difenoconazole WG and 1% phenazino-1-carboxylic acid SC can be used as alternative fungicides.