Human diseases have been one of the most commonly targeted for therapeutics based on RNA. Polymeric nanoparticles either natural or synthetic have been used widely for the genes’ transport across cell membranes. CS has been widely used as a polycationic polymer for drug delivery. Cross-linkers play a vital part in the preparation of stable nanoparticles. The aim of the present study was to formulate siRNA-loaded CS- poly-D-glutamic acid sodium salt (PGA) nanoparticles using ionic gelation (adsorption method) and assess it considering the physicochemical properties. The siRNA loaded CS-PGA nanoparticles were successfully prepared, and the results showed that the average diameter of nanoparticles was in the range from 400.0 ± 55.6 to 3575.5±71.2 by changing the CS concentration from 0.1 to 0.4% w/v. The zeta potential observed was in the range of +60.8 ± 1.1 to +38.2 ± 1.4 by changing the CS concentration from 0.1 to 0.4% w/v. siRNA-loaded CS-PGA nanoparticles showed aggregation and irregular morphology. All nanoparticles showed the high encapsulation efficiency in the range of 82% to 96%. The release profile for CS-PGA nanoparticles showed sustained release of siRNA. Based on the results, siRNA-loaded CS-PGA nanoparticles could be used for further optimization studies such as stability and steric hindrance.