A pair of primers were designed and synthesized according to the previously published sequence of fusion protein (F) gene of Newcastle disease virus (NDV) and used to amplify F gene by reverse-transcription polymerase chain reaction (RT-PCR) from the genomic RNA of a NDV strain JS5 isolated from goose. The PCR product was identified by sequencing. Then recombinant eukaryotic expression vector pVAX1-F was constructed through inserting F gene into MCS of pVAX1. The recombinant plasmid pVAX1-F was transfected in COS-7 cells, and identified for the transient expression of F gene by indirect immunofluorescent assay. Finally, the recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was screened and designated as SL7207 (pVAX1-F). It was verified that SL7207 (pVAX1-F) as the oral NDV DNA vaccine was safe for chickens after oral immunization at dosage of 10(10) CFU or below. 1-day-old commercial ISA brown chickens were immunized orally with SL7207 (pVAX1-F) at two different dosages (10(9) CFU and 10(8) CFU) on day 1, 14 and 28. On day 7 after the last immunization, no significant difference was observed in the body weight between these two groups (p > 0.05), and also no significant difference between those two groups and negative control group (p > 0.05). Since there were maternal antibodies, high ELISA titers of serum antibodies against NDV were detected in the chickens of all groups on day 14. However, the levels of serum antibodies were decreased in the chickens of all groups on day 28, but the anti-NDV antibody response detected in the sera of chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU were increased and significantly higher than the response induced by immunization with SL7207 (pVAX1) on day 35 (p < 0.05). Intestinal mucosal immune response was observed in chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU or 10(8) CFU. The high ELISA titers of antibodies against NDV in small intestinal mucosal samples from immunized chickens were on day 28 and 35. After challenged intranasally with virulent NDV strain F48E8, the chickens immunized with SL7207 (pVAX1-F) at the dosage of 10(9) CFU could be protected with the protective rate of 77.27%, significantly higher than those with SL7207 (pVAX1) (p < 0.05). In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium was safe and has good immunogenicity for chickens. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of ND in the poultry.