Cotton fibers with ultra-high purity cellulose are ideal raw materials for producing nanocellulose. However, the strong hydrogen bond and high crystallinity of cotton fibers affect the dissociation of cotton fibers to prepare nanocellulose. The structures of two kinds of cotton fibers (CM and XM) in different growth stages from 10 to 50 days post-anthesis (dpa) were studied by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). In the process of cotton fiber growth, the deposition rate of cellulose macromolecules firstly increased and then stabilized. Then, the surface morphology, the chemical composition, and the crystal structure of the nanocellulose prepared from cotton fibers with different growth stages by deep eutectic solvent, a green solvent, were characterized by Transmission Electron Microscope (TEM), scanning electron microscopy (SEM) analysis, XRD, and Thermo Gravimetry (TG). The growth time of cotton fibers affected the properties of prepared nanocellulose, and nanocellulose obtained from cotton fibers at about 30 dpa had less energy consumption, higher yield, and milder reaction conditions.