In this study, pCAGG-ChIL2 plasmid DNA containing the chicken interleukin-2 (ChIL-2) gene was used to prepare DNA-chitosan nanoparticles (CNPs). The CNPs prepared were spherical, with mean diameters between 100 and 200 nm, have a positive surface charge, and could protect DNA against DNase I degradation. The CNPs prepared were successfully used to transfect the Df-1 cell line with almost no cytotoxicity. CNPs prepared at an amino group to phosphate group ratio (N/P ratio) of 16 provided the highest transfection efficiency (1.1%) in medium with a pH of 6.5. When pCAGG-ChIL2 CNPs were administered to chickens simultaneously with a DNA vaccine against Newcastle disease virus (NDV), haemagglutination inhibition antibody titers and serum interferon-γ (IFN-γ) levels were significantly higher than in chickens immunised with the NDV DNA vaccine alone (p < 0.05). The results demonstrate that pCAGG-ChIL2 CNPs improve DNA vaccine-elicited immunity against NDV challenge.