According to the lack of a comprehensive non-clinical study for evaluating the national diphtheria-tetanus (DT) vaccine, we developed an experimental DT vaccine for comparing immunogenicity and efficacy of the DT vaccine with the international standard vaccine. Experimental vaccines were formulated as an adjuvant and non-adjuvant models using diphtheria and tetanus antigens with aluminum sulfate (alum) adjuvant. We used a unique formulation of the vaccine that was associated with greater antigen uptake on the alum gel. The quality control tests including identity (flocculation), adsorption, and potency tests have been performed to evaluate the potency of the vaccines. The pattern of the humoral immune response was calculated and plotted by an indirect ELISA method following immunization in mice model. The immunogenicity of the diphtheria-tetanus vaccine was investigated with serum neutralization (SN) and ELISA tests. Our findings indicated that the experimental DT vaccine was capable of inducing a long-term humoral immune response in mice and could induce a strong humoral response after the first injection. These results demonstrated the efficacy of the above vaccine in monovalent and divalent forms. A significant difference was observed between vaccines formulated with and without adjuvant. These findings indicated adjuvants can stimulate stronger humoral immunity against the target antigen and can be effective in prolonging immunity. To conclude, the experimental DT vaccine has efficacy and is capable to stimulate humoral immune responses.