Compared with the traditional surfactant-stabilized emulsions, Pickering emulsions stabilized by solid particles have the advantages of strong interfacial stability, versatility, and low toxicity. A number of cases have demonstrated the potential of Pickering emulsions in biomedical applications. Submicron Pickering emulsion has a larger specific surface area and more efficient delivery efficiency than large-sized Pickering emulsions, which is expected to further expand the advantages of Pickering emulsions in the biomedicine fields. However, the size of Pickering emulsions is determined by many factors such as particle properties, characteristics of oil and water phase. And it is difficult to arrange the rigid particles in a limited and small oil–water interface. The above reasons increase the difficulty of preparing the submicron Pickering emulsions. The purpose of this study is to prepare stable submicron-sized Pickering emulsion by utilizing the natural polysaccharide with good biocompatibility-cellulose nanocrystals (CNCs) as particle emulsifier, squalene as oil phase. The effects of preparation conditions such as particle concentration, oil–water ratio, aqueous phase, ultrasonic time and frequency on size distribution and stability of Pickering emulsions were investigated. Finally, the submicron-sized Pickering emulsion (CNCs-PE) with good storage stability and centrifugal resistance was prepared, and the average size of CNCs-PE was about 638.7?8.40 nm. The confocal laser scanning microscopic (CLSM) images revealed that Pickering emulsion was formed successfully by the adsorption of CNCs on the oil–water interface. The cytotoxicity of CNCs and CNCs stabilized Pickering emulsion (CNCs-PE) was evaluated using CCK-8 method, and it showed that there was no significant loss of cell viability. In addition, the vaccine formulation was prepared by absorbing antigen protein-OVA. The adsorption efficiency of OVA was about 80%, and the histological micrographs of the intramuscular injection site section also showed the injection safety. It is expected to expand the applications of submicron-sized Pickering emulsion based on CNCs in biomedicine fields.