Because of the high sensitivity and specificity and unparalleled ability to quantitatively analyze tissue microenvironments, fluorescence lifetime imaging microscopy (FLIM) is widely used in the field of life sciences. However, the use of FLIM in live imaging is hindered by its slow imaging speed. Recent advancements in optoelectronics and artificial intelligence technologies have ushered in a new era for in vivo FLIM. This review introduces ways to improve the imaging speed of the time-domain and frequency-domain FLIM techniques through hardware optimization and algorithmic improvements as well as via advancements in fundamental biomedical research and clinical diagnostics. Finally, we explore the future directions of the in vivo FLIM technology.