The invention belongs to the field of plant fungous disease pathogen culture, and more specifically discloses a making method and a using method of Fusarium head blight grain fungus strains. The making method is used for solving problems in the conventional fungus inoculation identification method that the method is complex, long time storage of fungus liquid is difficult to realize, and fungus inoculation success rate is low. The making method comprises following steps: grains with husk are immersed in 5% mung bean water to maintain 60% water holding capacity, and are packaged with bags; compacting is carried out, and the bags are sealed to prepare fungus tubes; the fungus tubes are subjected to high temperature high pressure sterilization, and are cooled to room temperature in the air; the two ends of each fungus tube are inoculated with fungus, and culture is carried out at a dark constant-temperature ventilative high humidity environment, and then obtained fungus strains are transferred into ventilative paper bags, are dispersed, are subjected to airing at cold and ventilative places, and are sealed in ziplock bags for low temperature storage in dark. The using method comprisesfollowing steps: activity is detected; after heading before flowering, basal spikelet side floret glumelle is inoculated with the fungus strains, packaging moisture retention is carried out until lemma becomes brown and a packaging layer is removed, and then mist moisture retention is carried out for 28d at high humidity. The advantages are that: 1, the obtained Fusarium head blight grain fungusstrains can be stored for a long term; 2, Fusarium head blight grain fungus strains prepared via one time of preparation can be used in a plurality of times, using amount is low, and transportation isconvenient; and 3, fungus inoculation method is simple, and is high in efficiency, incidence of disease is high, and disease resistance identification results are accurate.