The invention provides a method for rapidly separating endophytic fungi of tobacco. The method comprises steps of three-step disinfecting of tissue surface of a tobacco plant, slicing culture, separation and purification of endophytic fungal strains, and preservation. The method adopts the tissue separation method to separate and culture endophytic fungi from roots, stems and leaves of the tobaccoplants, and can better explore the biodiversity of endophytic fungi of the tobacco; at the same time, according to the tissue characteristics of the tobacco materials, the appropriate surface disinfection time and a disinfectant are selected, and three-step disinfection method of 75% of ethanol (1min), 2.5% of sodium hypochlorite (30s) and 75% of ethanol (1min) is established, and the separationeffect of endophytic fungi can be ensured to the greatest extent. The method has the characteristics and significance of establishing a more perfect separation technology method for endophytic fungi of the tobacco, and realizes more rapid and accurate separation; The method has important significance for the full utilization of tobacco endophytic fungi resources as well as the healthy and sustainable development of tobacco agricultural production.