Foot-and-mouth disease (FMD) is a devastating animal disease. Differentiation of infected from vaccinated animals (DIVA) is very important for confirming suspected cases, evaluating the prevalence of infection, certifying animals for trade and controlling the disease. In this study, a competitive chemiluminescence immunoassay (3B-cCLIA) for DIVA was developed for the rapid detection of antibodies against non-structural proteins (NSPs) in different species of livestock animals using monoclonal antibody 9E2 as a competitive antibody that recognizes NSP 3B, which only needs to be washed once and takes 15 min. The cut-off (50%), diagnosis sensitivity (97.20%, 95.71%, and 96.15%) and diagnosis specificity (99.51%, 99.43%, and 98.36) of the assay were estimated by testing a panel of known background sera from swine, cattle and sheep. The accuracy rate of 3B-cCLIA was also validated and compared with that of two commercial diagnostic kits. The early diagnostic performance showed that antibodies to NSPs occurred later (approximately 1–2 days) than antibodies to structural proteins. Furthermore, NSP antibodies present in animals vaccinated multiple times (false-positive), especially in cattle and sheep, were confirmed, and the false-positive rate increased with the number of vaccinations. These results indicated that 3B-cCLIA is suitable to rapidly detect antibodies against FMDV NSP 3B in a wide range of species for DIVA.