Abstract: In order to make full use of the by-products of industrial production of ginkgo, this study used ginkgo nut shell as raw materials and adopt ultrasonic-assisted sulfuric acid hydrolysis to prepare ginkgo nut shell nanocrystalline cellulose (nanocrystalline cellulose isolated from ginkgo nut shell, NCC-GNS). The effects of three factors (e.g. sulfuric acid mass fraction, reaction temperature, reaction time) on nanocellulose yield were investigated by single-factor tests, and orthogonal experiments were used to optimize them to obtain the best preparation conditions for NCC-GNS. Taking nanocrystalline cellulose (nanocrystalline cellulose, NCC) prepared by conventional sulfuric acid hydrolysis (without ultrasound assistance) as a control, analyzed the impact of ultrasound-assisted processing on NCC-GNS through scanning electron microscopy (SEM), transmission electron microscopy (TEM), Zeta potential and dynamic light scattering (DLS), X-ray diffraction (x-ray diffraction, XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), etc. The results showed that the optimal conditions for preparing NCC-GNS were sulfuric acid mass fraction of 48%, reaction temperature of 60 °C, reaction time of 25 min under the condition of ultrasonic power of 120 W. The NCC-GNS yield under optimal conditions was 37.01%. The NCC-GNS prepared by ultrasonic-assisted and conventional sulfuric acid hydrolysis methods were long rods with no significant difference in size. The length and diameter of the NCC-GNS prepared by ultrasonic-assisted were relatively concentrated with a length of 80~180 nm and a diameter of 3.5~5.5 nm. The crystallinity of NCC-GNS prepared by ultrasound was 88%, which was higher than 75% of conventional sulfuric acid hydrolysis. The NCC-GNS prepared by the two methods had lower Zeta potential and good thermal stability. In summary, the yield of NCC-GNS prepared by ultrasonic-assisted sulfuric acid hydrolysis was high, and the obtained NCC-GNS had high crystallinity and good thermal stability, which was expected to have better applications in the field of biomass composite materials.