<p>G-quadruplex/hemin DNAzyme, a versatile tool for biosensing, is challenged by its low peroxidase-mimic activities. The addition of NH₄⁺ may offer an efficient approach to improve its activity. However, the detailed impact of NH₄⁺ on its catalytic activity remains unclear, confusing the selection of appropriate DNAzymes for biosensing applications. Here, we conducted a comprehensive examination of the influence of NH₄⁺ on G-quadruplex/hemin DNAzyme. The results revealed that all DNAzymes with different G-quadruplex topologies exhibited increased catalytic activities in the presence of NH₄⁺ relative to K⁺, followed by the subsequent activity order: parallel &gt; hybrid &gt; antiparallel. Further investigations indicated that the increased catalytic activity can be ascribed to the increased stability of the G-quadruplex/hemin complex, elevated reaction velocity, and improved substrate affinity. Leveraging the significant disparity in enzymatic activity between parallel and antiparallel G-quadruplexes, an allosteric sensor based on the Pb²⁺-induced topological conformation was developed for sensitive detection of Pb²⁺ in the NH₄⁺-boosted G-quadruplex/hemin DNAzyme system (LOD, 1.56 nM), indicating potential for practical applications. Our discovery improves the understanding of NH₄⁺-boosted G-quadruplex/hemin DNAzyme and may facilitate the development of biosensors.</p> Graphical Abstract <p></p>