We report here the study of two-photon induced luminescence of 11-mercaptoundecanoic acid functionalized quantum-sized Au nanodots (11MUA-Au nanodots). The X-ray absorption near-edge structure (XANES) measurement at the gold LIII edge indicates that the oxidation state of the surface of 11MUA-Au nanodots is best described as Au+. The two-photon absorption (TPA) property of 11MUA-Au nanodots was then investigated via an open aperture Z-scan method. Together with a precise extinction coefficient (i.e., one-photon absorption) extracted from inductively coupled plasma mass spectrometry, the TPA cross section, σ, was deduced to be 8761 ± 175 GM (800 nm) for 11MUA-Au nanodots (diameter 1.33 ± 0.12) in water. Upon dextran encapsulation, 11MUA-Au nanodots was proved to be highly water-soluble, stable, and low in toxicity, which demonstrated its power in two-photon imaging toward human mesenchymal stem cells (hMSCs) in vitro.