As a new type of fluorescent nanomaterial, gold nanoclusters (AuNCs) are molecular aggregates composed of several to about one hundred gold atoms. Due to its simple preparation, excellent optical properties and low toxicity, AuNCs have been widely used in the field of biosensing in recent years. This review first briefly introduces the commonly used template method and its advantages for the preparation of AuNCs using sulfhydryl compounds, dendrimer, peptides and proteins, oligonucleotide DNA, etc. as templates. This review describes the UV absorption, fluorescence and electrochemical properties of AuNCs. After that, the application of fluorescent AuNCs-based biosensors in the detection of biomacromolecules and small molecules is summarized. Finally, the challenges of AuNCs in the field of biosensing are analyzed and their applications are prospected.