Animal growth traits are directly linked with the economics of livestock species. A somatostatin DNA vaccine has been developed to improve the growth of animals. However, the growth-promoting effect is still unsatisfying. The current study aimed to evaluate the effect of a novel eukaryotic dual expression vaccine known as pIRES-S/CST14-S/2SS, which encodes the genes obtained by fusing somatostatin (SS) and cortistatin (CST) into hepatitis B surface antigen (HBsAg). After transfection into GH3 cells with pIRES-S/CST14-S/2SS, green fluorescence signals were observed by fluorescence microscopy, suggesting the effective expression of CST and SS in GH3 cells using the IRES elements. Subsequently, both GH and PRL levels were found to be significantly lower in pIRES-S/CST14-S/2SS-treated cells as compared to the control group (p < 0.05). Furthermore, the antibody level, hormone secretion, and weight gain in the mice injected with novel recombinant plasmids were also evaluated. The anti-SS antibodies were detectable in all vaccine treated groups, resulting in significantly higher levels of GH secretion (p < 0.05). It is worth mentioning that pIRES-S/CST14-S/2SS (10 μg/100 μL) vaccinated mice exhibited a higher body weight gain in the second immunization period. This study increases the understanding of the relationship between somatostatin and cortistatin, and may help to develop an effective growth-promoting DNA vaccine in animals.