Fluorescence Lifetime Imaging Microscopy (FLIM) is an advanced tool that enables the description of exponential decay rate distribution of fluorescent molecules in the samples. This technique has been broadly used in biomedicine, material science, chemistry, and other related research fields, due to its ability to illustrate both localization of specific fluorophores and fluorophores' local microenvironment, and it is superior to fluorescence intensity based imaging. However, the FLIM imaging speed is inherently limited due to the long exponential decay collecting process, which may not be proper for monitoring fast dynamic biological processes in tissue, not to mention at single protein level. Excellent fluorescence labeling techniques, advanced imaging techniques and e±cient analytical tools together enable faster FLIM imaging. As the application of FLIM in biological field progresses, new requirements for FLIM technique are proposed, such as protein–protein interaction, label-free detection, deep tissue imaging, and so on.