The discovery and development of genetically-encoded fluorescent proteins (FPs) have revolutionized biology and medicine by allowing the visualization of molecular localization in live cells and animals. A general and high-efficiency strategy for developing and optimizing FRET-based biosensor can transform the field of biosensor development and allow the detailed investigation of different intracellular molecules, for the purpose of understanding and treating human diseases. Fyn kinase plays crucial roles in regulating a variety of important biological functions, including T cell receptor signaling, learnig and memory, as well as cell adhesion, mechanotransduction, and prostate cancer development. Therefore, we propose to develop a systematic strategy based on directed evolution to develop and optimize a Fyn FRET biosensor for the visualization of the subcellular Fyn activity in live cells, utilizing a high-efficiency FRET pair. Accordingly, two specific aims are proposed: (1) Optimize a substrate peptide sequence for Fyn kinase; (2) Develop an optimized SH2 domain and Fyn FRET biosensor. This systematic and directed evolution method can be readily extended for the development of, in principle, any FP-based genetically-encoded FRET biosensor. The information obtained should also advance our in-depth understanding of the molecular mechanism underlying the Fyn regulation. Hence, by integrating cutting-edge technologies in molecular engineering and live cell imaging, the success of the proposal should serve as a starting point to revolutionize the development of FRET biosensors and have transformative impact on biological studies in general.