Abstract: In this study, cellulose nanocrystals were prepared from microcrystalline cellulose.It used enzymatic hydrolysis and high-pressure homogenization to process cellulose nanocrystals which was firstly pretreated by ultrafine grinding. Besides, the structure and physico-chemical properties of cellulose nanocrystals was investigated. The obtained cellulose nanocrystals were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and thermo gravimetric analysis.The results showed that ultrafine grinding made its particles and shape tend to uniform and did not change the property of the microcrystalline cellulose.The nanocrystals were rod-shaped, with a diameter ranging from 15 to 40 nm.During the preparation of cellulose nanocrystals, the cellulose structure had not been destroyed. The molecular cellulose type I crystalline structure remained intact while the crystallinity was 58.1%. The initial decomposition temperature of cellulose nanocrystals was lower than microcrystalline cellulose.The lost weight of cellulose nanocrystals was82.9% when the temperature reached 500 ℃.The cellulose nanocrystals that prepared from enzymatic hydrolysis and high-pressure homogenization have potential application in biodegradable composites.