The SARS-CoV-2 pandemic has led to major advances in mRNA vaccine development. This study aimed to develop a cationic lipid nanoparticle (LNP) coated mRNA vaccine targeting the receptor binding domain (RBD) against the SARS-CoV-2 virus and adding valine to the N-terminal end to increase its stability, and to comprehensively characterize the physical, thermal, microscopic and biological properties of vaccine. The vaccine was designed in silico and valine-added S-RBD molecule was generated by IVT. The LNP coated mRNA vaccine was evaluated by spectroscopic (UV–vis, FT-IR, Raman), thermodynamic (TGA/DSC), microscopic (STEM, AFM), size/potential (DLS) and surface tension analysis methods. The developed vaccine was analyzed for cytotoxicity (MTT) and pro/anti-inflammatory gene expression (qPCR). Spectroscopic analyses revealed distinct RNA and lipid peaks. Thermodynamic analyses revealed complete mass loss for mRNA and LNP at 136.2 °C and 85.7 °C, respectively. STEM analysis revealed that mRNA was uniformly coated with LNP, and its size was < 100 nm, and Ra/Rz values were 26.56/34.29 nm, respectively. Zeta potentials ranged from 0 to − 30 mV, and surface tensions were 33.8–38.0 mN/m, respectively. MTT analysis showed that the vaccine was not cytotoxic. qPCR analysis, significant increases were detected in IFNA1, IFNB1 and TNF gene expressions, but no significant increase was detected in IL6. Bioinformatic analyses revealed increased stability in mRNA structure and more robust pseudoknot formations after valine addition. Our study is the first study in which a uniquely designed valine-added mRNA vaccine coated with LNP targeting the RBD antigen of SARS-CoV-2 was developed and comprehensively characterized.