Time-of-flight secondary-ion mass spectrometry (ToF-SIMS) is an advanced surface-sensitive technique that can provide both spectral and imaging information about materials. Recently, ToF-SIMS has been used for advanced studies of lignocellulosic biomass. In the current article, the application of ToF-SIMS to the characterization of the surface chemical composition and distribution of biomass components in lignocelluloses is reviewed. Moreover, extended applications of ToF-SIMS in the study of pretreatments, modification of biomaterials, and enzyme activity of lignocellulosic materials are presented and discussed. Sample preparation prior to ToF-SIMS analysis and subsequent interpretation of results is a critical factor in ensuring reliable results. The focus of this review is to give a comprehensive understanding of and offer new hints about the effects of processing conditions on the surface chemistry of lignocellulosic biomass.