We reported a method for in situ peeling force measurement of one-dimensional nanostructures using a dual-probe nanotweezer, which is developed on the principle of force microscopy. Benefiting from capabilities of image scanning and accurate force sensing, the nanotweezer is capable of positioning one-dimensional nanostructures deposited on a surface and then performing in situ peeling tests with pick-and-place operations at different peeling locations of interest along a selected nanostructure. In experiments, nanoscale peeling of silicon nanowires (SiNWs) on a silicon substrate has been studied. Peeling locations at the end and in the middle of the SiNW were tested and the results indicate that approximate peeling energies are needed.