The development of a tumor-targeted immunotherapy is highly required. The most advanced application is the use of CD19 chimeric antigen receptor (CAR)T (CAR-T) cells to B cell malignancies, but there are still side effects including potential carcinogenicity of lentiviral or retroviral insertion into the host cell genome. Here, we developed a nonviral aptamer-T cell targeted strategy for tumor therapy. Tumor cells surface-specific ssDNA aptamers were conjugated to CD3+T cells (aptamer-T cells) using N-azidomannosamine (ManNAz) sugar metabolic cell labeling and click chemistry. We found that the aptamer-T cells could specifically target and bind to tumor cells (such as SGC-7901 gastric cancer cell and CT26 colon carcinoma cell) in vitro and in mice after adoptively transfer in. Aptamer-T cells led to significant regression in tumor volume due to being enriched at tumor microenvironment and producing strong cytotoxicity activities of CD3+T cells with enhanced perforin, granzyme B, CD107a, CD69, and FasL expression. Moreover, aptamer-T displayed even stronger antitumor effects than an anti-PD1 immune-checkpoint monoclonal antibody (mAb) treatment in mice and combination with anti-PD1 yielded synergic antitumor effects. This study uncovers the strong potential of the adoptive nonviral aptamer-T cell strategy as a feasible and efficacious approach for tumor-targeted immunotherapy application.