The invention discloses an aureobasidium pullulans OF-01 strain (CGMCC No.7609) (China General Microbiological Culture Collection Center Number 7609), a method for preparing a tobacco endogenous derived spice by using the strain, and an application of the strain in a tobacco additive. A tobacco leaf fragment is used as a raw material, the aureobasidium pullulans OF-01 strain is used for fermentation, a plurality of products, such as extracellular polysaccharides (pullulan), enzymes, antibiotics, single-cell protein, volatile aroma components and the like, are generated, and the fermented tobacco leaf fragment is extracted and condensed to prepare the tobacco endogenous derived spice, therefore, the application of the spice and flavor products which are not derived from the tobacco in cigarette is reduced to achieve the re-utilization of the tobacco leaf fragment. The analysis on the aroma components proves that the spice comprises a plurality of types of tobacco aroma components and other derived aroma components. The result of a flavoring smoke panel test proves that the spice can be used for obviously increasing cigarette flavor, causing the smoke pure and mild, reducing the irritation, covering the offensive odor, and improving the cigarette smell. The spice has the characteristics of low cost, rich aroma components, safety to people and livestock and the like, therefore, the spice can be used in the production of the cigarette.