Synthetic nonionic surfactant vesicles (niosomes) are a colloidal system with closed bilayer structures, displaying distinct advantages in stability and cost compared with liposomes. In this article, polysorbate cationic niosomes (PCNs) were developed as gene carriers. The PCNs comprised nonionic surfactants (i.e., polysorbates) and a cationic cholesterol, and were synthesized using a film hydration method. The niosomes thus prepared possessed a regular morphology, and a particle size of 100 ∼ 200 nm, and a zeta potential of +30 ∼ 45 mV. The PCNs showed great physical stability over the course of 4 weeks at room temperature. The binding capacity of PCNs toward oligodeoxynucleotides (ODN) was assessed by a gel retardation approach, which demonstrated that the ionic complexes were formed when ± charge ratio reached to 4 or greater. Gene transfer study showed that the PCNs exhibited a high efficiency in mediating cellular uptake and transferred DNA expression. Based on these findings, PCNs may offer the potential to function as an effective gene delivery system.