In this review, we aim to present an overview of how small-molecular fluorescent substrate probes for studying enzymatic functions are developed and how they are used in biological applications, under the following four headings: (1) History of Visual Detection of Enzymatic Activities, (2) Strategies to Design Fluorescent Substrate Probes to Measure Enzymatic Activities, (3) Development of Fluorescent Substrate Probes Suitable for Biological Studies, and (4) Biological Applications of Fluorescent Substrate Probes for Studying Enzymes.