Herein, a label-free fluorescent signal amplification system based on a catalyzed hairpin assembly (CHA) is reported. In this system, two hairpin probes, H1 and H2, were well-designed in which G-quadruplex sequences were integrated into H2. The CHA reaction was triggered by target/trigger DNA and G-quadruplex sequences were released, which can bind the fluorescent amyloid dye thioflavin T (ThT) to provide fluorescence signals. At the same time, target/trigger DNA was released from the product of the CHA reaction (H1-H2), which continued to initiate the next CHA cycle, and the signal was eventually amplified. This signal amplification approach has been successfully used to develop a label-free fluorescent sensing platform for sensitive detection of human immunodeficiency virus (HIV) DNA and Pb2+.