Objective:To prepare chitosan nanoparticles as gene carrier of multiepitope DNA vaccine,and explore the loading capability and its protection ability to plasmid. Methods:The chitosan nanoparticles were prepared with ionic cross-linking method. The encapsulation rate was determined by UV spectrophotometer. The binding ability and protective effect of plasmid pIRES-TH-FL were evaluated by agarose gel electrophoresis analysis. The size,morphology and distribution were observed by transmission electron microscope. The diameter and zeta potential were measured by zetasizer. The pIRES-TH-FL-chitosan nanoparticles were transfected into 293T cells in order to examine their expression. Results:The encapsulation efficiency of pIRES-TH-FL-chitosan nanoparticles was 99.28% by UV spectrophotometer. Agarose gel electrophoresis showed that chitosan nanoparticles can effectively combined with plasmid pIRES-TH-FL and protected it from nuclease degradation.The mean diameter of pIRES-TH-FL-chitosan nanoparticles was about 300 nm and the mean zeta potential was 32.4mV.Western blot assay result confirmed that the DNA-chitosan nanoparticles can be expressed in 293T cells. Conclusion:pIRES-TH-FL-chitosan nanoparticles of homogeneous diameter were prepared and successfully expressed in vitro.