Interfacial nanopores enable real-time, single-cell monitoring by detecting subtle changes in ion flow or secretion profiles. We have developed a novel method to form nanopores at the interface between a (soft) substrate and a capillary, allowing the creation of pores at arbitrary locations – such as adjacent to a single cell – and with tunable pore sizes. Because the pore forms at the interface of two materials without any microfabrication techniques, it is called an interfacial pore. A micromanipulator precisely controls the capillary’s position relative to the surface to define the pore size, enabling sub-nanometer resolution. The ability to fine-tune the pore size enables selective detection of biomolecules according to their physical properties (e.g., charge and size). Placing these interfacial nanopores near cells makes it possible to monitor a range of activities, including changes in secretion profiles during cell activation, ion channel dynamics in the cell membrane, and shifts in extracellular potential in neurons during firing.