DNA vaccines are capable of eliciting both humoral as well as cellular immune responses. Liposomes have been widely employed for DNA delivery through topical route; however, they suffer from certain drawbacks like higher cost and instability. In present study, non-ionic surfactant based vesicles (niosomes) for topical DNA delivery have been developed. DNA encoding hepatitis B surface antigen (HBsAg) was encapsulated in niosomes. Niosomes composed of span 85 and cholesterol as constitutive lipids were prepared by reverse phase evaporation method. Prepared niosomes were characterized for their size, shape and entrapment efficiency. The immune stimulating activity was studied by measuring serum anti-HBsAg titer and cyokines level (IL-2 and IFN-gamma) following topical application of niosomes in Balb/c mice and results were compared with naked DNA and liposomes encapsulated DNA applied topically as well as naked DNA and pure recombinant HBsAg administered intramuscularly. It was observed that topical niosomes elicited a comparable serum antibody titer and endogenous cytokines levels as compared to intramuscular recombinant HBsAg and topical liposomes. The study signifies the potential of niosomes as DNA vaccine carriers for effective topical immunization. The proposed system is simple, stable and cost effective compared to liposomes.