Piezoelectric nanowire based nanogenerator is a promising technology to harvest ambient mechanical energy. It is essential to experimentally quantify the strain–piezopotential relationship on nanowires for the development of high-output nanogenerators. In this paper, 3D Kelvin probe microscopy (3DKPM) is applied to precisely mapping the piezopotential along a bent ZnO microwire (MW). In order to remove the charge screening effect and recover the actual piezopotential generated by the MW, an external DC bias was applied along the axial direction of the bent MW. This external field drove charged species in and outside of the MW to the two oppositely-biased ends, respectively, and thus minimized the screening effect. We also developed a numerical method to calculate the strain distribution along the bent ZnO MW based on its scanning electron microscopy (SEM) image, with which the strain–piezopotential relationship was obtained. The overall theoretical and experimental relationships showed a good match, indicating 3DKPM under biased condition can be an effective approach for quantifying piezopotential from strained nanomaterials. The detected piezopotential is independent of screening charge and external screening effect, and is not affected by the sharp topography variation along the edge of wires. It could serve as an important methodology for revealing nanoscale piezoelectric and flexoelectric properties.