Abstract: The 5′ untranslated region (5′UTR) of mRNA plays a crucial role in its expression. Using the enhanced green fluorescent protein gene (EGFP) as a reporter gene to detect the regulation of mRNA expression by 5′UTR is a well-established experimental method. This study aims to investigate the impact of the 5′UTR of herpes simplex virus 1 (HSV-1) immediate-early genes on the translation of EGFP mRNA when used as its 5′UTR. To achieve this, the 5′UTRs of four immediate-early genes and the strongly expressive human β-globin gene (hBg) 5′UTR were inserted at the 5′ end of the reporter gene EGFP. By observing the intensity of green fluorescence, the effect of different immediate-early gene 5′UTRs on mRNA translation was studied. The results showed that, when delivered into 293T cells and mice using mRNA transfection reagents and nanoliposome particles, the expression level of EGFP with the RL2 5′UTR was similar to that of the hBg 5′UTR, while the expression levels of RS1, US1, and US12 were significantly weaker than that of the hBg 5′UTR. Therefore, the RL2 gene's 5′UTR has potential as a 5′UTR for mRNA vaccines.