<p>A novel “signal-on” fluorescence sensing strategy was carried out based on GR5 DNAzyme and covalent organic framework (COF) to detect lead ions (Pb²⁺). The COF was synthesized at room temperature, with excellent structural stability and high fluorescence quenching efficiency, enabling low background signal and reliable detection. The GR5 DNAzyme that responds to Pb²⁺ contains a large single-stranded loop structure, which can be adsorbed onto the COF, resulting in fluorescence quenching of the attached FAM fluorophore. In the presence of Pb²⁺, the GR5 DNAzmye is specifically activated and cleaved, releasing a short FAM-labeled fragment of 5 bases. Because this short fragment has weak affinity for the COF, it is released from the COF surface, leading to fluorescent recovery. The sensor based on COF/DNA enzyme shows high selectivity and sensitivity, with a detection limit of 0.067 nM. In addition, the method has been successfully applied to the detection of Pb²⁺ in real water samples, with recoveries ranging from 89.58% to 112.70%. This work provides a simple and sensitive platform for Pb²⁺ detection and further demonstrates the potential of COFs as high-efficient fluorescence quenchers in functional nucleic acid-based biosensing.</p> Graphical Abstract <p></p>