Gold nanocages with localized surface plasmon resonance peaks in the near-infrared region exhibited a broad two-photon photoluminescence band extending from 450 to 650 nm when excited by a Ti:sapphire laser at 800 nm. The bright luminescence makes it possible to explore the use of Au nanocages as a new class of optical imaging agents for two-photon microscopy. In this work, we have demonstrated the use of two-photon microscopy as a convenient tool to directly examine the uptake of antibody-conjugated and PEGylated Au nanocages by U87MGwtEGFR cells. We have also correlated the results from two-photon microscopy with the data obtained by inductively coupled plasma mass spectrometry. Combined together, these results indicate that the antibody-conjugated Au nanocages were attached to the surface of the cells through antibody-antigen binding and then internalized into the cells via receptor-mediated endocytosis. The cellular uptake process was dependent on a number of parameters, including incubation time, incubation temperature, size of the Au nanocages, and the number of antibodies immobilized on each nanocage.