Engineered nanopipette tools have recently emerged as a powerful approach for electrochemical nanosensing, which has major implications in both fundamental biological research and biomedical applications. Herein, we describe a generic method of target-triggered assembly of aptamers in a nanopipette for nanosensing, which is exemplified by sensitive and rapid electrochemical single-cell analysis of adenosine triphosphate (ATP), a ubiquitous energy source in life and important signaling molecules in many physiological processes. Specifically, a layer of thiolated aptamers is immobilized onto a Au-coated interior wall of a nanopipette tip. With backfilled pairing aptamers, the engineered nanopipette is then used for probing intracellular ATP via the ATP-dependent linkage of the split aptamers. Due to the higher surface charge density from the aptamer assembly, the nanosensor would exhibit an enhanced rectification signal. Besides, this ATP-responsive nanopipette tool possesses excellent selectivity and stability as well as high recyclability. This work provides a practical single-cell nanosensor capable of intracellular ATP analysis. More generally, integrated with other split recognition elements, the proposed mechanism could serve as a viable basis for addressing many other important biological species.