Brachytherapy has been clinically used for the treatment of malignant solid tumors. However, the classic therapeutic radioactive 125I seed must be surgically implanted directly into tumors. To avoid the surgery and prevent irrational radioactive distribution, radioiodine-loaded nanomaterials are ever-developing for brachytherapy. Hence, it is still a notable challenge to obtain an advanced material that simultaneously incorporates features of high radiolabeling rate, short labeling time, good radiolabeling stability, and long tumor retention time. Covalent organic frameworks (COFs), which are crystalline polymers with ordered pores, are widely applied in guest delivery of drugs based on their high porosity and modifiable skeleton. Herein, we developed a functionalized nanoscale PEG-COF-Ag material, which could rapidly capture radioiodine reaching a 94% radiolabeling yield in 30 s. In addition, more than 95% 125I was maintained after 24 h in PBS (phosphate-buffered saline) as well as in serum and over 90% for nearly 1 week. PEG-COF-Ag-125I (125I-COF) demonstrated excellent cancer cell killing performance in vitro, and further experiments in vivo revealed a long tumor retention time and effective tumor treatment during the radiotherapy. The results indicate that radioiodine-labeled PEG-COF-Ag could be potentially applied in brachytherapy with a promising therapeutic effect.