The mRNA vaccine is an innovative new platform that can be developed rapidly, offering a robust tool to managing infectious diseases like COVID-19. However, non-clinical evaluations are essential for safety use. In this study, the efficacy, general toxicity, and safety pharmacology of a novel multivalent mRNA-encapsulated lipid nanoparticle (LNP) vaccine (RGV-DO-003) were evaluated using a focus reduction neutralization test (FRNT), virus challenge test, single- and repeated-dose studies, immunogenicity tests, and neurobehavioral, body temperature, respiratory, and cardiovascular system assessments in experimental animals. The administration route was intramuscular, and the vaccine doses were 5 and 50 μg/head for each efficacy test and toxicity study. Neutralizing antibodies were observed in the vaccinated group through FRNT and virus challenge tests. Macroscopic observations revealed a tendency for the lung lesion area to decrease in the vaccinated group. In the general toxicity study, mild symptoms were observed at the injection site in the vaccine administration group. Immunogenicity analysis revealed an increase in the binding antibody titer in the vaccine-administered group. Safety pharmacology study revealed no significant toxicological changes associated with LNP or vaccine administration. Overall, the RGV-DO-003 mRNA vaccine is effective and safe under experimental conditions, suggesting it is a potential candidate for clinical trials.