Immunotherapy represents an intervention in managing a wide variety of pathological diseases, including cancer and auto-immune disorders. The newest form of immunotherapy is cell and gene therapy, wherein immune cells are modified to express specific molecules and are genetically altered to generate a tailored immune response for a specific disease. While some of the viral vectors are FDA-approved and used as delivery vehicles, non-viral techniques are widely being explored for a safe and effective way to deliver genetic material to cells for therapeutic effect. Directed towards advancing a non-viral transfection system for immunotherapy, this study focuses on developing chitosan (CS) nanoparticles loaded with plasmid DNA (pDNA) and demonstrating their efficacy through both in vitro and in vivo transfection studies. These chitosan nanoparticles are synthesized via ionotropic gelation. When complexed with plasmid, they were found to be spherical in shape (~150 nm) and positively charged (zeta potential > 20 mV) as analyzed through dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and scanning electron microscopy (SEM). They exhibit good stability even after ten days of storage at 4 °C. Furthermore, their ability to transfect various immune cells in vitro was assessed using the GFP (green fluorescent protein) encoding plasmid. In addition, these nanoparticles loaded with luciferase (Luc) plasmid were assessed for their biodistribution and in vivo transfection in mice. Our results demonstrate that an optimized concentration of nanoparticles can be used for effective genetic modification of multiple immune cells in vitro as well as in vivo. Overall, these chitosan plasmid polyplexes hold promise as effective carriers of nucleic acid vaccines for immunotherapeutic applications.