The study investigates the two-photon luminescence (TPL) properties of gold nanorods, which exhibit strong TPL intensities when excited at 830 nm. The TPL excitation spectrum overlaps with the longitudinal plasmon band, indicating plasmon-enhanced two-photon absorption. The TPL signal from a single nanorod is significantly brighter than that from a single rhodamine molecule. The polarization dependence of TPL is cos4, and the emission is essentially depolarized. These characteristics make gold nanorods suitable for multiphoton microscopy. In vivo imaging of single nanorods flowing through mouse ear blood vessels demonstrates the potential of gold nanorods as imaging agents. The intrinsic 3D spatial resolution and rapid clearance time of TPL signals suggest their suitability for cell-specific labeling in biological applications.The study investigates the two-photon luminescence (TPL) properties of gold nanorods, which exhibit strong TPL intensities when excited at 830 nm. The TPL excitation spectrum overlaps with the longitudinal plasmon band, indicating plasmon-enhanced two-photon absorption. The TPL signal from a single nanorod is significantly brighter than that from a single rhodamine molecule. The polarization dependence of TPL is cos4, and the emission is essentially depolarized. These characteristics make gold nanorods suitable for multiphoton microscopy. In vivo imaging of single nanorods flowing through mouse ear blood vessels demonstrates the potential of gold nanorods as imaging agents. The intrinsic 3D spatial resolution and rapid clearance time of TPL signals suggest their suitability for cell-specific labeling in biological applications.