In our previous study, we designed and evaluated the efficacy of six DNA vaccine candidates based on the E protein of Zika virus (ZIKV). To optimize the DNA vaccine, we inoculated C57BL/6 and IFNAR1 mice with the vaccine candidate expressing tandem repeated ZIKV envelope domain III (ED III × 3) doses; 50 μg by intramuscular (IM), jet injection (JET), or electroporation (EP) routes. Results showed that vaccination by all routes induced humoral and cellular immunity. Among them, EP induced robust ZIKV E specific-total IgG and neutralizing antibodies, as well as T cell responses. Additionally, EP showed superior protective efficacy against the ZIKV Brazil strain compared to the IM and JET routes. Finally, in the dose optimization test of EP route, cellular immunity of 50 μg was induced a significant level than other dose groups. These results showed that the EP delivery system enhanced the potential immunogenicity and protective efficacy of DNA vaccines.