Gold nanoparticles (AuNPs) with unique physical and chemical properties have become an integral part of research in nanoscience. In particular, the development of AuNP-based fluorescent oligonucleotide (AuFO) probes targeted at living systems through fluorescence are drawing increasing attention and have become a current hot topic in biomedical research. This review describes recent advances and developments in the use of AuFO probes from their preparation and features that allow the desired intracellular delivery to their applications in cellular imaging and drug delivery in living systems.