This chapter discusses the principles, techniques, and applications of chemical and biological sensors based on fluorescence lifetime measurement. Fluorescence lifetime, which is the duration that fluorescent molecules reside in the excited state before returning to the ground state, is highly sensitive to environmental changes and is independent of initial concentration, absorption, fluorescence intensity, and other factors, making it valuable in chemical analysis and biomedical research. The chapter begins by introducing the basic concepts and measurement methods of fluorescence lifetime, including time-domain and frequency-domain techniques. It then delves into the applications of fluorescence lifetime imaging microscopy (FLIM) in the field of biosensors, such as FRET-FLIM, pH, and temperature measurements, and other microenvironmental parameters. Additionally, the chapter covers the applications of FLIM in the field of chemical sensing, including microfluidics, chemical reactions, and material characterization. Finally, the chapter summarizes the advantages and challenges of FLIM technology and provides a perspective on its future development directions.