Abstract: Disclosed herein is an optimized CD99-targeting chimeric antigen receptor and application thereof, wherein a signal peptide, a single-chain antibody ScFv, strepII, a CD8 hinge, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB and CD3? chain are sequentially spliced in the chimeric antigen receptor is from the N-terminal to the C-terminal. The single-chain antibody ScFv can specifically recognize CD99 protein on the surface of tumor cells. The CD99-targeting chimeric antigen receptor is used to modify immune cells for the treatment of surface CD99-positive tumors.