The invention belongs to the technical field of traditional Chinese medicine fermentation, and provides astragalus membranaceus fermentation liquor as well as a preparation method and application thereof. The preparation process comprises the following steps of soaking astragalus membranaceus slices in water, decocting, and filtering to obtain medicine residues and first medicine liquid, adding water into the medicine residues, and decocting to obtain second medicine liquid, combining the first medicine liquid and the second medicine liquid, and concentrating to obtain an astragalus membranaceus water extract, mixing lactobacillus plantarum LZU-S-ZCJ with LZU-J-TSL6, and inoculating the mixture into the astragalus membranaceus water extract for fermentation, so as to obtain the astragalus membranaceus fermentation liquor. The astragalus membranaceus fermentation liquor obtained by the method disclosed by the invention can relieve the edema degree of mouse lungs, relieve the inflammatory cell infiltration state caused by lipopolysaccharide (LPS), reduce the accumulation of protein liquid in the mouse lungs, reduce the concentration of inflammatory factors in BALF of mice with acute lung injury, regulate the frontal intestinal flora of the mice with acute lung injury and the like, can be used for treating acute lung injury, and has a good effect of relieving mouse acute lung injury.