Abstract: DNA vaccines carrying multiple copies of repetitive sequences show great potential in aquatic animal disease prevention and control, but their large-scale production process remains a key bottleneck hindering industrialization. To achieve high-yield, high-quality plasmid DNA, this study optimized the fermentation process for the recombinant E. coli strain Stbl3 harboring plasmid DNA vaccines with multiple copies of repetitive sequences. Shaker flask experiments identified glucose as the optimal carbon source at a concentration of 5 g/L. In a 5 L fermenter scale, parameters including inoculation timing, inoculum volume, dissolved oxygen level, feeding strategy, and temperature induction conditions were optimized. The final process conditions were established as follows: inoculation at 4.5% in mid-log phase, maintaining dissolved oxygen at 30%, and setting induction temperature at 42 ℃ during the late fermentation stage. This process was successfully validated in a 30 L fermenter, achieving a final cell density of OD₆₀₀ 42.3 and plasmid yield to 178.6 mg/L, of which the supercoiled fraction exceeded 80% and repetitive sequence fragments kept integrity. Animal studies confirmed the DNA vaccine's favorable biosafety profile, with challenge immunity trials achieving over 70% protection rates. Collectively, the fermentation process optimization for multicopy repetitive sequence plasmids using recombinant E. coli Stbl3 provides an applied foundation for industrial-scale development of aquatic DNA vaccines.