In recent years, the potential value of nonstructural protein (NSP) 2C was well documented for distinguishing foot-and-mouth disease virus (FMDV) in infected animals and vaccinated animals. In order to develop a more sensitive approach to detect natural infected FMDV while there is no interact with vaccinated FMDV, we incorporated a major epitope region of 2C with whole 3AB coding region within NSP and expressed in Escherichia coli. We got a 47.6 kD fusion protein named 2C￠3AB. The product showed a specific reactivity with FMDV from serum of infected animal by using Western blotting analysis. This suggests that this protein could be applied to distinguish infected FMDV and vaccinated FMDV. We further compared 2C￠3AB protein with 3ABC fusion protein, another available protein used for detecting infected FMDV, using indirect ELISA assay. The results showed that 2C￠3AB-ELISA had higher sensitivity than that of 3ABC-ELISA for distinguishing infected FMDV and vaccinated FMDV of sera from epidemic region. Therefore, this recombinant protein 2C￠3AB is a good candidate protein to develop more sensitive method to differentiate infected FMDV and vaccinated FMDV from vaccinated animals. This finding will increase our capability to check the infectious virus carrier and finally improve FMDV infection control.