The invention belongs to the field of natural medicine and medicinal chemistry, and relates to a preparation method and uses of a brefeldin A derivative with melphalan-based chlormethine linked at site 4, specifically to a preparation method for introducing a DNA alkylating agent melphalan derivative at the 4-OH site of brefeldin A, and applications in preparation of antitumor drugs. According tothe present invention, the brefeldin A derivative with melphalan-based chlormethine linked at site 4, and the pharmaceutically acceptable salt have a structure represented by a general formula I, wherein n is defined in the claim and specification.