Investigating biological and synthetic nanoscopic species in liquids, at the ultimate resolution of single entity, is important in diverse fields1-5. Progress has been made6-10, but significant barriers need to be overcome such as the need for intense fields, the lack of versatility in operating conditions and the limited functionality in solutions of high ionic strength for biological applications. Here, we demonstrate switchable electrokinetic nanovalving able to confine and guide single nano-objects, including macromolecules, with sizes down to around 10 nanometres, in a lab-on-chip environment. The nanovalves are based on spatiotemporal tailoring of the potential energy landscape of nano-objects using an electric field, modulated collaboratively by wall nanotopography and by embedded electrodes in a nanochannel system. We combine nanovalves to isolate single entities from an ensemble, and demonstrate their guiding, confining, releasing and sorting. We show on-demand motion control of single immunoglobulin G molecules, quantum dots, adenoviruses, lipid vesicles, dielectric and metallic particles, suspended in electrolytes with a broad range of ionic strengths, up to biological levels. Such systems can enable nanofluidic, large-scale integration and individual handling of multiple entities in applications ranging from single species characterization and screening to in situ chemical or biochemical synthesis in continuous on-chip processes.