The authors demonstrate the use of gold nanorods as bright contrast agents for two-photon luminescence (TPL) imaging of cancer cells in a three-dimensional tissue phantom, achieving depths of up to 75 μm. The TPL intensity from gold nanorod-labeled cancer cells is significantly brighter than the two-photon autofluorescence (TPAF) emission from unlabeled cells at 760 nm excitation. Gold nanorods exhibit strong signal, resistance to photobleaching, chemical stability, ease of synthesis, and biocompatibility, making them suitable for TPL imaging of epithelial cancer. The study also explores the performance of nanorods as contrast agents for deep-tissue imaging, showing that TPL imaging of labeled cancer cells requires less excitation power compared to TPAF imaging. The optimal excitation wavelength for both gold nanorods and cellular autofluorescence is 760 nm, allowing for a fair comparison of emission intensities. The research highlights the potential of gold nanorods in expanding the capabilities of TPL imaging to enable non-invasive, three-dimensional imaging of molecular signatures in tissue.The authors demonstrate the use of gold nanorods as bright contrast agents for two-photon luminescence (TPL) imaging of cancer cells in a three-dimensional tissue phantom, achieving depths of up to 75 μm. The TPL intensity from gold nanorod-labeled cancer cells is significantly brighter than the two-photon autofluorescence (TPAF) emission from unlabeled cells at 760 nm excitation. Gold nanorods exhibit strong signal, resistance to photobleaching, chemical stability, ease of synthesis, and biocompatibility, making them suitable for TPL imaging of epithelial cancer. The study also explores the performance of nanorods as contrast agents for deep-tissue imaging, showing that TPL imaging of labeled cancer cells requires less excitation power compared to TPAF imaging. The optimal excitation wavelength for both gold nanorods and cellular autofluorescence is 760 nm, allowing for a fair comparison of emission intensities. The research highlights the potential of gold nanorods in expanding the capabilities of TPL imaging to enable non-invasive, three-dimensional imaging of molecular signatures in tissue.