Fluorescent biosensors are crucial experimental tools for live-cell imaging and the quantification of different biological analytes. Fluorescent protein (FP)-based biosensors are widely used for imaging applications in living systems. However, the use of FP-based biosensors is hindered by their large size, poor photostability, and laborious genetic manipulations required to improve their properties. Recently, semisynthetic fluorescent biosensors have been developed to address the limitations of FP-based biosensors using chemically modified fluorescent probes and self-labeling protein tag/peptide tags or DNA/RNA-based hybrid systems. Semisynthetic biosensors have unique advantages, as they can be easily modified using different probes. Moreover, the self-labeling protein tag, which labels synthetically developed ligands via covalent bonds, has immense potential for biosensor development. This review discusses the recent progress in different types of fluorescent biosensors for metabolites, protein aggregation and degradation, DNA methylation, endocytosis and exocytosis, membrane tension, and cellular viscosity. Here, we explain in detail the design strategy and working principle of these biosensors. The information presented will help the reader to create new biosensors using self-labeling protein tags for various applications.