Nanocrystalline cellulose (NCC) has attracted a significant interest in recent years as it is made from renewable resources and has many unique characteristics. In this paper, we prepared NCC from cotton pulp fiber by the method of sulfuric acid hydrolysis, with the objective of achieving a maximum yield. The yield and the particle size of NCC were measured as a function of sulfuric acid concentration, reaction temperature and hydrolysis time, and optimized by an orthogonal test. A high NCC yield of more than 60% was found when the sulfuric acid concentration is 64%, reaction temperature is 50°C and the hydrolysis time is 5h. The morphology and the size of NCC were characterized by scanning electron microscope (SEM) and particle size analyzer. The finding from this study should provide guidance in maximizing the yield of NCC from cotton pulps using acid hydrolysis.