Cellulose nanocrystals have been applied as reinforcing material in polymer matrices due to their excellent mechanical properties and the fact that they are derived from natural fibers, which are biodegradable in the environment and come from renewable sources. This paper studies the morphological, dimensional, chemical and surface characteristics of cellulose nanocrystals (CNC) isolated from cotton fibers pre-treated with sodium hydroxide (NaOH) 2% m/v focusing on hydrolysis time. Such characterization used transmission electron microscopy (TEM), X-ray diffractometry (XRD), Fourier-transform infrared spectroscopy (FTIR), thermogravimetry (TGA), zeta potential, dynamic light scattering (DLS) and energy-dispersive X-ray spectroscopy (EDS). The isolated cellulose nanocrystal yield was evaluated based on hydrolysis of the material with 65% sulfuric acid (m/m), at a temperature of 50 °C, acid:biomass ratio of 20 mL g-1 and hydrolysis times of 50, 70, 90, 110 and 130 min. It was found that treatment with 2% NaOH provided satisfactory accessibility to the fibers and the effect of hydrolysis time on CNC production showed a peak yield at 110 min, 33%. The CNC had negative surface charges, -30.0 to -53.3 mV, giving stability to the suspensions. CNC obtained under the conditions evaluated can be used in the composition of composites in various hydrophilic matrices because maximum degradation temperatures are above 200 °C and because they are stable and of uniform size. © 2019 Assoc. Tecnica Brasileira de Celulose e Papel. All rights reserved.