The invention relates to the technical field of litchi preservation, and particularly discloses a room-temperature litchi storage preservative, and a preparation method and an application of the preservative. The preservative mainly comprises carboxymethyl-chitosan, nisin, natamycin, tea polyphenol, epsilon-polylysine, citric acid and phytic acid; litchi fruit is soaked in a preservation solution, and then aired, so that the litchi fruit can be stored at a room temperature; the quality and low level browning of the litchi fruit can be kept for a long time at the room temperature. Litchi treated by the preservative has the characteristics that the preservative is good in preservation effect, safe, environment-friendly, simple to operate and convenient to use; the preservative is extensive in source, low in cost, edible, and easy to degrade; no pesticide residue risk exists; short-term preservation during room-temperature storage and room-temperature logistics can be achieved; the preservative is a novel preservation material with a great development potential.