As a new type of nanomaterials, the gold nanoclusters (AuNCs) perform many special physical and chemical properties, such as large Stokes shift, relatively simple preparation, good water solubility, low toxicity, and good biocompatibility, which make them show infinite potential in various fields, especially in cancer treatment. In recent years, great progress has been made in the preparation, functionalization, and biomedical applications of the AuNCs. In this article, the latest research progress and synthesis methods of the AuNCs have been summarized, emphasizing the preparation using the “bottom- up” synthesis strategy. Furthermore, we introduced the in vivo pharmacokinetic performance of the AuNCs. In the last part, we exemplified the applications of the AuNCs in biomedicine, including photothermal therapy (PTT), bioimaging, drug delivery, and radiotherapy sensitization, which further confirmed the great potential of the AuNCs in tumor treatment.