Chitosan nanoparticles were prepared by crosslinking chitosan (CS) with tripolyphosphate (TPP) solution using electrohydrodynamic spraying technique. The effects of CS and TPP concentration as well as electrical potential on particle size and shape were investigated. Appropriated formulations for preparing nanoparticles were chosen to encapsulate DNA. In vitro evaluation of the obtained nanoparticles as gene carrier such as entrapment efficiency and DNA release was performed. The results showed that 2 mg/ml TPP was dropped at 10 kV into 1 mg/ml CS (MW 20 kDa (F1) and 200 kDa (F2)) yielded the spherical shape and small particles of 227.67 and 240.33 nm, respectively. In DNA entrapment study, all formulations were tested by altering DNA loading to 10, 25 and 50 mg/g of CS. The results revealed that F1 with initial DNA 10 mg/g of CS showed the highest entrapment efficiency of 95.31%. While F2 with initial DNA of 25 mg/g of CS showed the highest entrapment efficiency of 89.16%. The DNA release study from CS nanoparticles indicated that the increasing of DNA amount slowed down the release rate. F1 and F2 with the initial DNA of 10 mg/g of CS had faster release rate than those with 25 and 50 mg/g of CS. It can be concluded that F1 yielded the nanoparticles with the smallest size, high DNA entrapment efficiency and enabled DNA sustained release.