Characterization techniques of molecular interactions are powerful methods to elucidate cell biological events, understand the pathogenesis of diseases, and assist drug development. In recent years, the characterization techniques of molecular interactions have been developing rapidly and have been constantly moving toward high sensitivity, high throughput, extremely short time, and extremely low detection limits. This review summarizes the principles, characteristics, and latest application progress of 4 common optical detection-based characterization techniques of molecular interactions, including surface plasmon resonance, biolayer interferometry, back-scattering interferometry, and microscale thermophoresis, so as to provide a reference for the selection of characterization techniques of molecular interactions.