Abstract: Nanosized single crystal cellulose is often called whisker. Nano-whisker or nanofibrils can be prepared from various sources, such as natural fibers, agricultural by-products, and marine animals. Nano-whiskers are characterized by excellent mechanical properties, high specific surface area, high length-diameter ratio, environmental friendliness, and low cost. Because of these properties, nano-whiskers are used in many fields and attract growing attention by researchers in the world. In China, the agricultural product processing industry yields abundant cellulose waste. Thus, it becomes meaningful to find out a better resolution to 'agricultural cellulose waste'. In this study, nano-cellulose was prepared by a sulphuric acid hydrolysis method and with shells of sunflower seeds as raw material. The effects of four factors (e.g. acid hydrolysis temperature, sulfuric acid concentration, acid hydrolysis time, and liquid to material) on nano-cellulose yield were investigated by single-factor tests. The preliminary results showed that the optimal conditions were: acid hydrolysis temperature at 40℃; sulfuric acid concentration at 60%; hydrolysis time at 90 min; liquid to material ratio at 12:1. The results of single-factor tests showed that the nano-crystalline cellulose (NCC) yield was maximized to 26.72% at the acid hydrolysis temperature 40℃. The yield of NCC was maximized to 29.89% at the sulfuric acid concentration of 60%, to 30.09% at the acid hydrolysis time of 90 min, or to 25.33% at the liquid to material of 12:1. Based on the single-factor test, the process parameters were optimized by a response surface method. A quadratic polynomial model was built, and then its effectiveness and the interactions between different factors were analyzed. Also the microstructure of the nano-cellulose as-prepared was investigated by transmission electron microscopy (TEM), particle size distribution, Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). Results showed that the importance degrees of all factors on yield of NCC rank as follows: Acid hydrolysis temperature > acid hydrolysis time > liquid to material > sulfuric acid concentration. The optimal process conditions were as follows: acid hydrolysis temperature at 42℃, acid hydrolysis time at 83.71 min, sulfuric acid concentration at 59.97%, and liquid to material ratio at 12.33:1. Under these conditions, the highest yield of NCC was 31.67%, which was validated to be 31.31%. SEM and particle size distribution analysis showed that the NCC from shells of sunflower seeds was rod-like and partially aggregated. The diameter was measured to be 10 - 30 nm, and length was 150 - 300 nm. FT-IR showed that NCC still possessed the basic chemical structure of cellulose and did not have functional group variation. XRD showed that NCC possess a typical cellulose I crystal structure and their crystallinity degree was obviously higher the crystallinity degree. This study provided some reference for the comprehensive utilization of sunflower seeds.