The efficacy of chimeric antigen receptor (CAR)-T cell therapy relies on the single-chain variable fragment (scFv) domain which directly engages with target antigens on tumor cells and subsequently initiates CAR activation in T cells. However, conventional in vitro scFv screening methods for CAR-T cell therapy, which primarily access binding affinity to purified target proteins, often fail to translate into optimal therapeutic outcomes. In this study, we introduce an innovative scFv screening system based on fluorescence resonance energy transfer (FRET). This FRET-based CAR biosensor is designed to display FRET signals when the scFv binds to target antigens on tumor cells and initiates the CAR activation. For fine-tuning the scFv affinity of mesothelin (MSLN)-targeting CARs, we generated the biosensors containing scFv libraries with varying affinities, and live-cell FRET measurements identified a novel scFv sequence with enhanced cytotoxicity against tumor cells. This novel CAR showed superior immunological synapse formation and minimal CAR internalization. Therefore, the FRET-based CAR screening system is a powerful tool to optimize scFv affinity for maximizing efficacy of CAR-T cell therapy.