Cellulose nanocrystals (CNCs) have attracted greatly attention due to their excellent properties. As one of the methods to prepare CNCs, enzymatic enzyme-mediated methods are environment friendly and with expected lower capital compared to traditional processes, but not thought highly of because of the low yield and efficiency. Although particle morphology is considered to be a crucial parameter, the research on the shape-dependent properties of spherical CNCs has been limited to date. In this article, the spherical CNCs with high yield were obtained by enzymatic hydrolysis and mechanical homogenization of eucalyptus pulp and microcrystalline cellulose. By means of field emission scanning electron microscope, atomic force microscopy, ionic chromatograph, X-ray diffraction, Fourier transform infrared spectroscopy, dynamic light scattering, thermogravimetric analysis and rheology, the effects of types of raw materials, enzymatic hydrolysis conditions and homogenization times on the preparation of spherical CNCs were systematically studied. The results showed that spherical CNCs can be obtained from incompletely enzymatically hydrolyzed rod-like nanocellulose by high-pressure homogenization and the yield can reach 83.26% (eucalyptus pulp) and 75.50% (microcrystalline cellulose), which was much higher than that of spherical CNCs isolated by enzymatic hydrolysis alone. Fourier transform infrared spectroscopy and X-ray diffraction proved that the crystal phase of the spherical CNCs did not change, but the changing trend of crystallinity was different. The crystallinity of using eucalyptus pulp as the raw material decreased while the crystallinity of using microcrystalline cellulose increased. Compared with microcrystalline cellulose, the spherical CNCs obtained from eucalyptus pulp were smaller in diameter (10–20 nm) and the suspension was more transparent and stable. On the contrary, the thermogravimetric analysis showed that the spherical CNCs from microcrystalline cellulose had better thermal stability than one from eucalyptus pulp, and the temperature of maximum weight loss rate was 338°C. The rheology depicted that the spherical CNCs colloidal suspension was shear thinner and had a lower viscosity than the rod-shaped and fibrillar nanocellulose. Our results reveal that the spherical CNCs with high yield were obtained by using the green method to grade treatment cellulose, and the similarities and differences between nanocellulose prepared from natural fibers and commercial microcrystalline cellulose were compared.