Lignocellulosic nanofibers derived from tobacco stalk can have countless applications in polymers composites, textile, cosmetics, and pharmaceuticals. Thus, it is important to evaluate biomass characteristics such as the presence of nicotine. In this study, nanofibers were obtained by mechanical fibrillation while cellulose content (0.5 and 2.0%) and drying methods were varied. Nanofibers were characterized by thin layer chromatography, 1H NMR, morphological analysis, α-cellulose content, Fourier transform infrared spectroscopy, X-ray diffraction and thermal analysis. Results demonstrate the absence of nicotine in tobacco stalk. The grinding mill process was efficient to produce by freeze-drying, nanofibers with fiber’s mean diameter of ~30 nm. Solid concentrations can influence the diameter of obtained fibers. Thermal stability increased and crystallinity decreased when alkali treatment was applied. The characterization techniques applied enable the evaluation of tobacco stalk and expanded its application to pharmaceutics.