<p>Lignocellulosic biomass is a renewable carbon source that could help replacing fossil carbon feedstocks which cause many ecological concerns. However, to improve its bioconversion, the complex microstructure and chemistry of biomass needs thorough characterization. Emerging techniques like Fluorescence Lifetime Imaging Microscopy are particularly promising and this review aims to cover all aspects related to the use of lifetime microscopy for lignocellulosic biomass analysis. First, the mechanisms involved in fluorescence emission and atomistic properties influencing fluorescence lifetime are detailed. Then the three main instrumentations of lifetime microscopy are compared and the decay fitting function of fluorescence lifetime is presented. Numerous examples exposing the relevance of fluorescence lifetime imaging microscopy for biomass analysis are provided. Lifetime microscopy allows for cellulose, hemicelluloses, and lignins differential localization and syringyl / guaiacyl lignin ratio mapping. Fluorescence lifetime imaging microscopy can also provide insights on the effects of pretreatment and hydrolysis on the microstructure and chemistry of lignocellulosic biomass. Additionally, lifetime microscopy can inform on growth conditions like geographical origin or reaction wood formation as a response to gravitropic perturbations. Also, Förster Resonance Energy Transfer, being able to explore lignocellulosic biomass’s interactions with molecular probes, can be based on fluorescence imaging as well. Finally, other fluorescence-lifetime-related techniques having the potential to be implemented on lignocellulosic biomass are discussed.</p>