Gene therapy is considered the most advanced method for treating various diseases. Effective gene delivery requires overcoming intracellular barriers and developing cost-effective, efficient gene carriers for clinical applications. Both viral and non-viral systems have been explored as gene delivery vectors. Viral systems exhibit high transfection efficiency; however, their clinical application is restricted due to potential toxicity and immunogenicity concerns. Conversely, non-viral systems require high in vivo stability, ease of fabrication, and modification for enhanced therapeutic efficacy. Nanoparticles have demonstrated excellent properties for gene delivery among non-viral vectors, including targeted tissue or cell-specific delivery, protection against nuclease degradation, improved deoxyribonucleic acid (DNA) stability, and better transfection efficiency with enhanced safety profiles. Silk-based non-viral gene vectors are particularly promising due to their biodegradability, low toxicity, and functional versatility, which recombinant techniques can further enhance. The size and sequence of silk copolymers can be precisely controlled via genetic engineering for nanoparticle synthesis when coated with polyethyleneimine (PEI), have gained significant attention for targeted delivery of gene. This chapter highlights the preparation, processing, characterization, and genetic engineering strategies for designing and cloning silk fibroin nanoparticles for gene delivery and gene therapy applications.