Cell is the basic unit of structure and biological activities of animals and plants. An important feature of cellular processes is that the biochemical components involved in it have spatiotemporally interacted with each other. However, it is difficult to evaluate molecular interactions in living cells using traditional biochemical methods (e. g., yeast two-hybrid system, pull-down system, etc. ). The rapid development of optical technology provides new genetic research tools for studying the spatiotemporal dynamics of biomolecules in living cells. FRET-FLIM (Frster resonance energy transfer-fluorescence lifetime imaging microscopy) technology has unique advantages in real-time detection and analysis of conformational changes and dynamic interactions of macromolecules in living cells, such as achieving real-time “visualization” study of living cells with the high spatial and temporal resolution, obtaining more reliable results with high sensitivity, and accomplishing simple and fast analysis program based on brief mathematical operations etc. This paper outlined the theoretical backgrounds of FRET-FLIM, and then summarized the advantages and disadvantages of FRET-FLIM technique compared with traditional methods for protein interaction studies. In addition, we emphasized the recent studies and applications of FRET-FLIM in protein interaction, cell biology, and disease diagnosis. Finally, this paper prospected the future research trend of FRET-FLIM in the field of life sciences and provided a comprehensive and systematic reference for related research in the structure and processes of living cells.