The major difficulty in designing carrier system for gene delivery is finding a good compromise between structure, stability, and efficiency. Current carrier systems are limited in terms of release kinetics, toxicity, and overall capacity to carry biomolecules. The objectives of this study were to develop a process of producing biodegradable polymer-based hollow spheres and to characterize their potential use as carriers of plasmid DNA (pDNA) for gene delivery. The method developed involves coating a template with a polymer layer. After the removal of the template, a hollow core is obtained. Specifically, the production process begins with chitosan coating and is followed by polyglutamic acid cross-linking. The advantage of this production process is that it could be applied to a variety of polymeric systems. Because the hollow polymer structures can encapsulate a large variety of guest molecules, these spheres have significant potential for delivery. After physical characterization, the ability of the spheres to encapsulate and retain pDNA was investigated. The chitosan/polyglutamic acid spheres were used to encapsulate pDNA with up to 90% efficiency, and the encapsulated pDNA was retained for 3 days without degradation in 10% fetal bovine serum (FBS)-complemented media and released up to 30% after 3 days in the presence of the lysozyme, suggesting that these spheres represent an attractive method for gene delivery.