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A simple protease-free and signal-on electrochemical biosensor based on GR-5 DNAzyme and catalytic hairpin assembly (CHA) for sensitive lead ion (Pb2+) assay was constructed in this work. In the presence of Pb2+, GR-5 DNAzyme specifically reacted with Pb2+ and cleaved the substrate strand into two free DNA fragments, and one of the two fragments then opened the hairpin capture DNA (Hc) that was assembled on the Au electrode surface and triggered the CHA reaction. Exploiting the CHA strategy, a large number of the hairpin signal DNA (Hs) labeled with two methylene blues (Mbs) were captured on the Au electrode surface and generated an efficient electron transfer, resulting in the dramatic increase of methylene blue current. In this method, Pb2+ could be detected quantitatively in the range of 4 × 10−11–3 × 10−6 M, and the detection limit was as low as 2.7 × 10−11 M (S/N = 3). Experimental results demonstrated that the biosensor was highly specific for Pb2+ and exhibited remarkable improvements of electrochemical analytical performance. This biosensor was also used for the analysis of Pb2+ in the serum sample spiked with Pb2+, and the obtained result in good agreement with the correct values. The excellent performance of the biosensor shows its promising potential for the clinical diagnosis of lead poisoning.