Abstract: An indirect enzyme-linked immunosorbent assay for differentiating infected animals from vaccinated animals was established by using foot-and-mouth disease virus (FMDV) nonstructural protein (NSP) 3ABC antigen,which was expressed in E.coli. Serial experiments were performed using sera from cattle. A formula {value=（ODsam－ODneg）÷（ODpos－ODneg）}was developed to calculate sample values, critical values were determined by optical density (OD) of naive cattle sera, and values below 0.2 were considered as negative, values between 0.2 and 0.3 as ambiguous and values above 0.3 as positive. The sensitivity of the assay was 100%,as all the experimental postinfection sera gave values greater than 0.3. In contrast, 97.06% of sera from vaccinated animals were negative. A high degree of specificity was also confirmed by the finding that 98.39% of sera from naive animals gave negative results. The 3ABC specific antibodies were detectable at least 1 year postinfection. The described 3ABC-I-ELISA can differentiate infected from vaccinated animals and can perform in large scale serological surveys.