Zinc is a common redox element and plays a vital role in immune response, catalysis, structure, and signal regulation. Tracing intracellular Zn dynamics is, therefore, crucial for understanding physiological processes. Since a glass nanopipette is a good sensing platform for single-cell detection with little damage to the cell membrane, in this study, we developed DNAzyme-functionalized glass nanopipettes (D-GNs) for single-cell Zn detection based on the catalytic activity of Zn-dependent DNAzymes. In the presence of Zn, DNAzyme-modified gold nanoparticles (D-AuNPs) were released from the inner wall of the nanopipette, leading to a change in ionic current rectification. D-GNs exhibited good sensing performance in vitro and can be applied to effectively detect Zn in HeLa cells under electrical stimulation. This functional nanopipette-based platform offers a new strategy for probing intracellular ion variations and evaluating the physiological state of individual cells.