The survival rate for patients with relapsed and refractory acute myeloid leukaemia (R/R-AML) remains poor, and treatment is challenging. Chimeric antigen receptor T cells (CAR-T cells) have been widely used for haematologic malignancies. Current CAR-T therapies for acute myeloid leukaemia mostly target myeloid-lineage antigens, such as CD123 and CD33, which may be associated with potential haematopoietic toxicity. As a lineage-specific receptor, CD7 is expressed in acute myeloid leukaemia cells and T cells but is not expressed in myeloid cells. Therefore, the use of CD7 CAR-T cells for R/R-AML needs to be further explored. In this report, immunohistochemistry and flow cytometry were used to analyse CD7 expression in clinical samples from R/R-AML patients and healthy donors (HDs). We designed naturally selected CD7 CAR-T cells to analyse various functions and in vitro antileukaemic efficacy based on flow cytometry, and xenograft models were used to validate in vivo tumour dynamics. We