The development and utilization of lignocellulosic biomass resource becomes a major subject of widespread concern at home and abroad. To make better use of this resource and reduce cost, a thorough understanding of recalcitrance mechanism as well as chemical compositional and structural characteristics of lignocellulosic cell walls seems desiderated. With the property of fast testing, high sensitivity and simple sample preparation, FT-IR microspectroscopy can provide microanalysis and nondestructive test. It has been proved to accurately present compositional and structural information of lignocellulosic cell walls and be suitable for the study of topochemistry of main components at the cellular level. After a brief introduction of the principle of FT-IR microspectroscopy and its detailed experimental procedure, this review provides an overview on the feasibility of FTIR microspectroscopy in monitoring as well as evaluating the composition and structure of lignocellulosic cell walls from three aspects, including in situ distribution, changes after pretreatment and molecular orientation of the major components in cell walls. Some prospects, for example development of the magnification of the microscope and the spatial resolution of chemical imaging, combination with various analysis methods and instruments are also proposed. This technology can act as a new approach in characterizing lignocellulosic cell walls.