Autologous chimeric antigen receptor T (CAR-T) cell therapy has demonstrated efficacy in the treatment of acute myeloid leukemia (AML). Nevertheless, the intrinsic characteristics of autologous therapy, such as extended manufacturing timelines and patient-specific limitations, contribute to delays in treatment availability. More critically, relapse due to antigen escape following single-targeted CAR-T therapy constitutes a significant clinical obstacle. To address the dual challenges of delayed treatment accessibility and antigen escape relapse, this study proposes the development of universal tandem CAR-T cells. These cells, engineered to target CD123 and B7-H3 through clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology, represent an innovative therapeutic strategy for AML.In this study, an immune phage display nanobody library was developed for the purpose of screening CD123-specific nanobodies. The CRISPR/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system was utilized to disrupt the TRAC and B2M genes present in T cells, resulting in the generation of universal CD123/B7-H3 bispecific universal CAR-T (UCAR-T) cells. The efficacy of these dual-specific UCAR-T cells in combating tumors was subsequently assessed through in vitro and in vivo experiments.Through four rounds of panning against CD123 from an immunized camelid VHH library, we identified 21 antigen-specific nanobodies. Tandem bispecific UCAR-T engineered with these binders demonstrated CAR transduction efficiencies ranging from 82% to 87%. In vitro functional profiling revealed a significantly enhanced cytotoxicity of bispecific UCAR-Ts against CD123+/B7-H3+ AML cell lines when compared to single-target constructs, while effectively regulating the secretion of effector cytokines (IL-2, IFN-γ, TNF-α). In AML xenograft models, treatment with bispecific UCAR-T notably inhibited tumor progression, extended the survival of tumor-bearing mice with recurrence-free persistence throughout the observation period, and did not result in significant body weight loss or cytokine release syndrome.The findings of the study address the issue of tumor antigen evasion in the treatment of AML, circumvent certain constraints associated with autologous CAR-T cell therapy, and offer novel insights and strategies for managing AML.Copyright © 2025 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.