Targeted radionuclide therapy (TRT) has been a promising strategy for cancer therapy, which can inhibit or kill cancer cells by selectively delivering radionuclide to target tissues. Herein, a legumain-targeted therapeutic agent [131I]MAAN was synthesized that was capable of condensation and self-assembly in response to legumain and glutathione (GSH) in tumor cells to enhance the therapeutic effect. This radiopharmaceutical has been acquired with a radiochemical yield (RCY) of 57.8 ± 5.3% and a high radiochemical purity (RCP) of more than 95%. In vitro cellular uptake assay suggested that [131I]MAAN had a 2-fold higher uptake in legumain-positive HCT116 cells than in the legumain-negative SKOV3 cells, revealing the specificity of the agent for legumain. Similarly, the in vitro cytotoxicity assay showed that [131I]MAAN possessed a more obvious killing effect on HCT116 cells. Cerenkov imaging with [131I]MAAN was carried out to generate clear tumor images. TRT with [131I]MAAN significantly inhibited the tumor growth and no obvious toxicity towards other normal organs was observed, indicating that it is a safe and effective treatment for cancers. Therefore, the agent [131I]MAAN holds great potential for the diagnosis and treatment of cancers with a high expression level of legumain.