A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring. Environmental ImplicationsThis work provided unique strategy for environmental Pb2+ detection based on tetrahedral DNA nanostructure-enhanced hyperbranched hybridization chain reaction. Pb2+ are highly toxic and persistent environmental contaminants, which are difficult to biodegrade and easy to be enriched in the environment (e.g., the soil and water) or living organisms via bioaccumulation and biomagnification effects of food chains. Even negligible levels of lead can do harm to the human health, resulting in impaired kidney function, blood disorders, and even death. Therefore, it is greatly significant for public health to develop simple, rapid and ultrasensitive techniques for Pb2+ detection in environmental samples.