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Background Solid tumors present a major challenge to CAR-T therapy due to antigen heterogeneity, immunosuppressive tumor microenvironments, and toxicity concerns. To overcome these barriers, we developed a novel dual-targeting tandem CAR-T cell therapy co-expressing anti-PD-L1 scFv and monobody-based EphA2 binding domains. The PD-L1-targeting domain was derived from scFvs with improved specificity over existing PD-L1 antibody-based CARs, while the EphA2-targeting domain was constructed using a high-affinity monobody selected from a yeast display library.Methods Four distinct tandem CAR constructs were generated using a second-generation CAR backbone: PD-L1-EphA2-CD28 (dual 1), EphA2-PD-L1-CD28 (dual 2), PD-L1-EphA2-4-1BB (dual 3), and EphA2-PD-L1-4-1BB (dual 4). Constructs were transduced into human T cells using lentiviral vectors and expression was confirmed by FACS. Functional characterization included in vitro cytolysis assays (RTCA, IncuCyte), 3D tumor spheroid assays, and in vivo xenograft models (NOG mice) using multiple human cancer cell lines expressing either PD-L1 or EphA2. Dose-dependent anti-tumor efficacy and safety profiles were assessed.Results Among the constructs, PD-L1-EphA2-CD28 tandem CAR-T cells demonstrated the highest cytotoxicity and functional activity. These CAR-T cells achieved near-complete lysis (~100%) of PD-L1+ or EphA2+ target cells across diverse solid tumor lines, including ovarian, pancreatic, gastric, lung, liver, and colorectal cancers. In vivo, PD-L1-EphA2-CD28 CAR-T cells showed potent tumor control in an ovarian cancer xenograft model even at low doses (2×10 5 cells/mouse), with no signs of toxicity, weight loss, or mortality. At high doses (1×107 cells/mouse), mice maintained 100% survival, indicating a favorable safety profile.Conclusions Tandem PD-L1-EphA2 CAR-T cells combining enhanced specificity, broad target range, and a co-stimulatory CD28 domain show potent anti-tumor efficacy with minimal toxicity in preclinical models. These findings support the clinical development of PD-L1-EphA2 tandem CAR-T therapy for treating solid tumors expressing either or both antigens, and demonstrate a promising strategy for overcoming antigen escape and improving safety in CAR-T applications for solid malignancies.