Abstract: In recent years, the potential value of nonstructural protein (NSP) 2C was well documented for distinguishing footandmouth disease virus (FMDV) infected animals from vaccinated animals. In this study, 2C gene of FMDV was cloned into baculovirus transfer vector pFastbacHTB, and then the recombinant vectors were transformed into DH10Bac which contain backbone Bacmid. The white colonies were selected and Bacmid2C recombinant vectors were used for transfection of Sf9 cells. After three times amplification of the viral stocks, the NSP2C was expressed in High Five cells. The SDSPAGE showed that a 38.93 kD fusion protein 2C was successfully expressed, and the products showed a specific reactivity with serum from FMDV infected animal using Westernblot analysis and DotELISA. An indirect ELISA based on fusion protein 2C was developed, the results showed that this 2CELISA not only could be applied to distinguish FMDV infected animals from vaccinated animals, but also could be used to detect early anti2C antibodies in cattle and pigs which had been infected with FMDV. The baculovirus expressed 2C appears to be a suitable antigen for the development of a reliable diagnostic test. This study make a good foundation for development of diagnosis method for detection of antibodies against multiple NSPs to discriminate the FMDV infected animals from vaccinated ones, and this will increase our capability to check the infectious virus carrier and finally improve FMDV infection control.