This study validated a new recombinant protein-based solid-phase competitive ELISA (r-SPCE) for detecting antibodies to Foot-and-Mouth disease virus type-O. The r-SPCE kit demonstrated high specificity (99.9%) and sensitivity (95.2%) compared to the virus neutralization test. It was able to correctly detect international reference sera for FMDV type-O as well as differentiate it from other FMDV serotypes. The r-SPCE also showed excellent reproducibility, stability, and was easy to use for screening large numbers of cattle and pig samples. Overall, the validated r-SPCE is a reliable alternative to the virus neutralization test for serological assessment of FMD