Nucleic acid vaccines play a pivotal role in the development of advancing vaccine technologies against infectious diseases. Despite their potential, the clinical application of nucleic acid vaccines still faces hurdles including limited transfection efficacy and suboptimal biocompatibility. Addressing these challenges, this study introduced a novel strategy utilizing the self-assembly of poly(β-amino ester) (PBAE) with nucleic acids to form PBAE nanoparticles (PBAE-NPs). PBAE-NPs were further modified with poly(glutamic acid) (PGA) to produce PGA modified PBAE nanoparticles (PPs) as potential nucleic acid delivery carriers. Comprehensive synthesis, structural characterization, and biological evaluation of PPs were performed. The characterization results demonstrated that PPs exhibited favorable biological characteristics, such as well-controlled particle size, low zeta potential, and exceptional serum stability. The lysosomal escape experiment results showed that PPs had good lysosomal escape ability endowed with PGA. Flow cytometry results indicated that PPs exhibited significantly enhanced nucleic acid transfection efficiency compared to PBAE-NPs. The CCK-8 results showed that PPs had good biocompatibility. Collectively, these results suggest that PPs could significantly improve nucleic acid transfection efficiency, thereby providing insight into the formulations design for nucleic acid vaccines.