The invention belongs to the technical field of genetic engineering antibodies, and in particular relates to a preparation method and application of CD24 (cluster of differentiation 24) antibody fusion protein. A CD24 humanized antibody hG7S is connected with MICA (major histocompatibility complex class I chain-related gene A) molecule extracellular 1 to 3 domains through a G4S flexible peptide by utilizing a genetic engineering technology; an hG7S-MICA is stably expressed through a mammal eukaryotic expression system; CD24 humanized antibody hG7S-MICA fusion protein can be combined with a CD24 molecule on the surface of a tumor cell and an activated receptor NKG2D on the surface of an NK cell at the same time, and an immunologic surveillance effect of the NK cell induced by an NKG2D way on a CD24+tumor cell is rebuilt; finally, the aim of activating an organism self-immune system to effectively kill the CD24+tumor cell is realized.