We herein describe a novel method to identify thyroid hormone (TH)/thyroid hormone receptor (TR) interaction, termed aptamer-assisted protein-induced fluorescence enhancement (AptPIFE). In this method, a detection probe consisting of an RNA strand incorporating TH-specific aptamer and a Cy3-labeled DNA strand holds TH in close proximity to Cy3. The corresponding TR then binds to the TH near Cy3, consequently stimulating Cy3 to emit a significantly enhanced fluorescence through PIFE phenomenon. Based on this simple yet efficient design principle, we successfully identified the interaction of TH with TR within 10 min, down to 0.37 pM with excellent specificity. The practical and robust applicability of this method was also successfully validated by properly screening TR antagonists and reliably quantifying TH present in real clinical serum samples from patients with hyperthyroidism and healthy volunteers.