Abstract: 【Objective】 To identify the species of a fungal totacco leaf spot disease and its biological characteristics in tobacco producing area of Hezhou，Guangxi，so as to provide theoretical reference for timely prevention and control and further research of the disease.【Method】The pathogen was isolated and purified from totacco leaf tissues with obvious symptom of tobacco leaf spot collected from the tobacco producing area of Hezhou，Guangxi by tissue separation method.And its pathogenicity was identified according to Koch’ s rule.Morphological analysis and multi-gene sequence analysis （ITS，LSU，TUB2，RPB2）were used to identify the pathogen species.Biological characteristics of the pathogens were determined by cross method.【Result】 Seven strains of fungi were isolated and purified，and according to Koch’ s rule，all the 7 strains were pathogenic fungi of tobacco variety K326.No significant difference in pathogenicity among the 7 strains was found.A phylogenetic tree was constructed based on ITS，LSU，TUB2 and RPB2 genes.The results showed that all the 7 strains were clustered with Stagonosporopsis vannaccii LFNOI48 and YTH-12，and the support rate was 100%.The results of biological characteristics showed that the optimum temperature for the mycelium growth of the 7 strains was 25-28℃；the optimum carbon source was soluble starch；the optimum nitrogen source was peptone and beef extract powder；and the optimum pH was 6-9；the optimum medium was potato glucose medium（PDA），oat medium（OA）and pumpkin juice medium（PA）.Mycelia grew the fastest under full light，and the lethal temperature was 50℃ for 10 min.【Conclusion】 S.vannaccii is the pathogen of tobacco leaf spot disease in Hezhou tobacco producing area of Guangxi.This pathogen grows in environment of weak acid to weak alkaline conditions，with sufficient light and rich nutrition.Tobacco leaf spot caused by S.vannaccii can be prevented and controlled by means of arranging planting density，strengthening field management，chemical and biological control，etc.