Abstract: In recent years, tobacco leaf mold has become a serious disease in tobacco producing areas of Yunnan Province. To clarify the pathogen of the disease, the pathogen was isolated, identified and its biological characteristics were also analyzed based on the Koch's postulations, morphology observation and molecular technology. The results showed that the aerial hyphae of the strain Rhi-1 is vigorous and loose, with its sporangium diameter being 53-123 μm and its sporangiospore diameter being 2.1-9.0 μm, both being spherical or oval. Combining with the 16s rDNA-ITS sequence analysis, the strain Rhi-1 was identified as Rhizopus oryzae. The highest temperature of spore germination is 44.6℃, lethal temperature of the spores is 54℃ in hot water bath for 10 min and lethal temperature of the mycelium is 70℃ in hot water bath for 30 min. The optimal pH value is 7.0 and the optimal medium is PSA. These results could provide a good basis for pathogenicity studies of the pathogen.