Background: Inefficient cellular uptake is a significant limitation to the efficacy of DNA vaccines. In this study, we introduce S-Cr9T, a stearyl-modified cell-penetrating peptide (CPP) designed to enhance DNA vaccine delivery by forming stable complexes with plasmid DNA, thereby protecting it from degradation and promoting efficient intracellular uptake. Methods and Results: In vitro studies showed that S-Cr9T significantly improved plasmid stability and transfection efficiency, with optimal performance at an N/P ratio of 0.25. High-content imaging revealed that the S-Cr9T–plasmid complex stably adhered to the cell membrane, leading to enhanced plasmid uptake and transfection. In vivo, S-Cr9T significantly increased antigen expression and triggered a robust immune response, including a threefold increase in IFN-γ secretion and several hundred-fold increases in antibody levels compared to control groups. Conclusions: These findings underscore the potential of S-Cr9T to enhance DNA vaccine efficacy, offering a promising platform for advanced gene therapy and vaccination strategies.