Abstract: Cellulose nanocrystals(CNC) were prepared from microcrystalline cellulose(MCC) by sulfuric acid hydrolysis. The effects of sulfuric acid mass fraction, reaction temperature and reaction time on the yield and average particle size of cellulose nanocrystals were studied by single factor method combined with orthogonal test. The performance of CNC was characterized by scanning electron microscopy(SEM), atomic force microscopy(AFM), X-ray diffractometer(XRD) and nano-laser particle size analyzer, and the mechanism of acid hydrolysis to prepare CNC was revealed. The results showed that the optimum process parameters of CNC preparation were sulfuric mass fraction 64%, reaction temperature 45 ℃ and reaction time 90 min. Under these conditions, the yield of CNC was 24.6% and the particle size was 204.8 nm. The CNC aqueous suspension was a kind of stable light blue colloidal state, and its microscopic morphology was relatively regular, which was short rod-shaped, with diameter of about 10-20 nm and length between 150-300 nm; XRD results showed that the crystal form of CNC was cellulose type Ⅰ, and the crystallinity was 80.2%.