Abstract: Disclosed are a hexavalent norovirus VLP vaccine formulation and a preparation method thereof. The formulation comprises six proteins having amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, and SEQ ID No. 12. A Pichia pastoris expression system was used to develop the hexavalent norovirus vaccine. Sequencing, digestion, and exogenous gene expression detection were conducted to identify recombinant vectors and select high-expression strains. VLPs were purified by sucrose density gradient centrifugation and identified by electron microscopy, with the results demonstrating that the obtained candidate hexavalent vaccine VLPs had a purity of over 95% and each VLP was intact in structure and uniform in size, indicating structural integrity and stable performance of the VLPs. The hexavalent VLP vaccine induced long-lasting and high-titer antibody responses and generated specific blocking antibodies and T cell immune responses.