Due to the unique advantages of two-photon technology and time-resolved imaging technology in the biomedical field, attention has been paid to them. Gold clusters possess excellent physicochemical properties and low biotoxicity, which make them greatly advantageous in biological imaging, especially for in vivo animal imaging. A gold nanocluster was coupled with dihydrolipoic acid to obtain a functionalized nanoprobe; the material displayed significant features, including a large two-photon absorption cross-section (up to 1.59 × 10 5 GM) and prolonged fluorescence lifetime (>300 ns). The two-photon and time-resolution techniques were used to perform cell imaging and in vivo imaging.