The invention belongs to the field of food processing, and discloses a meaty seasoning with edible fungi flavor and a preparation method and application thereof. The meaty seasoning with the edible fungi flavor is prepared from edible fungi or edible fungi leftovers with different flavors and other food raw material components in the following mass ratio: based on 1,000 milliliters of flavoring liquid, 100 to 200 grams of mushroom or leftovers thereof, 50 to 100 grams of pleurotus eryngii or leftovers thereof, 50 to 100 grams of needle mushroom or leftovers thereof, 15 to 20 grams of xylose, 20 to 40 grams of glucose, 5 to 10 grams of salt, 2 to 3 grams of glycine, 2 to 3 grams of alanine, 5 to 10 grams of cysteine, 3 to 5 grams of thiamin hydrochloride and the balance of water. The preparation method provided by the invention is simple and feasible, and prepares the meaty fragrant seasoning with the edible fungi flavor. The meaty seasoning with the edible fungi flavor can be used forpreparing flavoring.