Science, medicine, clinical diagnostics, biotechnologies, and environmental technologies all require methods that are highly selective, sensitive, rapid, and reliable for identifying key ingredients or metabolites. These methods determine product quality or serve as markers for diseases, physiological states, or environmental safety. A wide range of dangerous pollutants, including heavy and transition metals, volatile toxicants, pesticides, plasticizers, pharmaceuticals, and natural and synthetic hormones, must be tested in various environmental media to predict potential hazards for humans and nature and to evaluate the effectiveness of wastewater treatment. Many physicochemical analytical approaches have been developed; however, they are not suitable for the high-throughput monitoring of chemicals in air, soil, aquatic, and biological samples. Biosensors are the most promising tools for these purposes. While enzymes have traditionally been used as biocatalytic elements in most biosensors, the development of microbial cell sensors has gained significant momentum only during the last decades. Microbial sensors, particularly yeast-based systems, show great promise in analytical practice. A microbial biosensor consists of a transducer combined with immobilized viable or nonviable microbial cells, offering an economical substitute for enzymes. The target analyte is typically either a substrate or an inhibitor of cell metabolism. We highlight the main achievements in developing analytical systems based on yeast cells and discuss their potential applications in clinical diagnostics, food control, environmental monitoring, and detoxification.