The invention discloses a preparation method and application of a tobacco extract. The preparation method comprises the following steps of using tobacco powder and/or tobacco waste as raw materials; using solvents for tobaccos as dispersing agents; performing uniform stirring; adding obtained mixed liquid into a sealing bag for sealing; putting the sealing bag in a liquid medium; performing ultra-high pressure treatment for 10 min to 60 min at the pressure of 200 MPa to 800 MPa; relieving the pressure, wherein the mass percentage of the raw materials to the dispersing agents is 0.1 percent to15 percent; performing high-pressure homogenization treatment on the mixed liquid subjected to ultra-high pressure treatment to obtain homogenization liquid, wherein the temperature of the high-pressure homogenization treatment is -20 DEG C to 0 DEG C, and the pressure of the high-pressure homogenization treatment is 10 MPa to 200 MPa; centrifuging the homogenization liquid; and collecting centrifuged supernatant liquid to obtain the tobacco extract. The conditions of the whole extraction process are mild; no heating step is performed; the process is simple; and the acquisition is easy. The tobacco extract well maintains the original taste and flavor of tobacco leaves, and is harmonious and natural with tobacco fragrance.