The BRAF V600E mutation induces sustained activation of the MEK-ERK signaling pathway, driving accelerated cell proliferation, enhanced migration, invasion, and resistance to apoptosis, which is recognized as a critical pathogenetic mechanism in refractory thyroid cancer. Small interfering RNA (siRNA) holds significant promise in cancer therapy due to its high specificity and silencing efficiency, ease of synthesis, and cost-effectiveness. However, its clinical application is limited by poor permeability and stability. DNA-assembled nanostructures, especially the tetrahedral DNA nanostructures (TDNs), are promising nanocarriers with preeminent biosafety, low biotoxicity, and high transport efficiency. In this study, we therefore synthesized a TDN-based BRAF V600EsiRNA delivery system (TDN-siBraf), which not only prolonged the half-life of siRNA, but also exhibited efficient siRNA delivery, excellent biocompatibility, and gene silencing efficiency. By establishing two distinct cell line models, a murine model and a notable in vitro patient-derived tumor organoids (PDTOs) model of refractory thyroid cancer, we demonstrated that TDN-siBraf could enhance targeted siBraf delivery and improve antitumor efficacy both in vivo and in vitro by inhibiting cancer cell proliferation and invasion while promoting apoptosis, with favorable biosafety and biocompatibility. Furthermore, treatment with the TDN-siBraf effectively inhibited the activation of the MEK-ERK signaling pathway, leading to mitochondrial dysfunction and elevated DNA damage, which ultimately culminated in cellular impairment. This study introduces a high-efficiency TDN-siBraf delivery system with prolonged siRNA half-life and enhanced antitumor activity in refractory thyroid cancer, offering a promising strategy with significant clinical translation potential.