We experimentally demonstrate a scheme to deterministically excite a three-dimensionally oriented electric dipole in a single Au nanosphere by using a tightly focused radially polarized beam whose focal field possesses polarization states along three-dimensional (3D) orientations owing to the spatial overlap between longitudinal and radial electric field components. Experiment observations indicate that the orientation of an excited dipole moment gradually changes from out-of-plane to in-plane when the nanosphere is moved away from the beam center, which is reconfirmed by full-wave simulations. Moreover, rigorous calculation based on Mie theory reveals that a reduced effective ambient permittivity accompanies the rotation of the dipole moment, leading to a blue-shifted and narrowed resonance peak. We envision that our results could find applications in detecting the 3D orientation of isolated molecules and benefit the fine manipulation of light–matter interactions at the single-molecule level.