The wide-field fluorescence microscopic imaging in the second near-infrared region (NIR-II, 900-1880 nm) is currently a hot spot for deep in vivo bioimaging, with great potential in both basic research and clinical applications. Compared to the visible (Vis, 360-760 nm) and the first near-infrared (NIR-I, 760-900 nm) region, NIR-II fluorescence in vivowide-field microscopic imaging provides higher spatial resolution and deeper tissue penetration. On the basis of NIR-II macroscopic imaging, the demand for deciphering of tissue microstructure forces the continuous development of fluorescent agents and the imaging system. NIR-II wide-field microscopic imaging technology has achieved a series of breakthroughs in biological applications such as accurate vascular microscopy, tumor analysis and accurate inflammation tracking. The research objects include rodents (such as mice and rats) and non-human primates (such as marmosets and macaques). With the breakthrough of instrument commercialization and localization as well as the increasing biocompatibility of fluorescent probes in the future, the application values of NIR-II fluorescence wide-field microscopic imaging will continue to upsurge. This article discusses the mechanism and advantages of NIR-II imaging first,and then, reviews the characteristics and history of NIR-II fluorescence wide-field microscopic imaging, as well as its latest exploration and prospects for in vivo imaging on different biological models. The purpose is to promote the further popularization of NIR-II fluorescence in vivowide-field microscopic imaging.