Recently, gold nanoclusters (AuNCs) have been widely used in biological applications due to their ultrasmall size, ranging within a few nanometers; large specific surface area; easy functionalization; unique fluorescence properties; and excellent conductivity. However, because they are unstable in solution, AuNCs require stabilization by using ligands such as dendrimers, peptides, DNA, and proteins. As a result, the properties of AuNCs and their formation are determined by the ligand, so the selection of the ligand is important. Of the many ligands implemented, enzyme-stabilized gold nanoclusters (enzyme-AuNCs) have attracted increasing attention for biosensor applications because of the excellent optical/electrochemical properties of AuNCs and the highly target-specific reactions of enzymes. In this review, we explore how enzyme-AuNCs are prepared, their properties, and the various types of enzyme-AuNC-based biosensors that use optical and electrochemical detection techniques. Finally, we discuss the current challenges and prospects of enzyme-AuNCs in biosensing applications. We expect this review to provide interdisciplinary knowledge about the application of enzyme-AuNC-based materials within the biomedical and environmental fields.