Numerous fundamental biological processes occur in subcellular locations or inside intracellular organelles. Nanoelectrodes allow precise and quantitative measurements of important biomolecules at the single living-cell level in real time. We are devoted to designing novel strategies for nanoelectrodes fabrication to explore unclear subcellular biological processes. We have developed carbon fiber nanoelectrodes for real-time monitoring of exocytotic dynamics inside single synapse and functionalized cylindrical nanowire electrodes for detection of intracellular events. Taking it further, the development of innovative nanoelectrode tactics and integration with other techniques will be conducive to deeper comprehension of subcellular processes.