Foot-and-mouth disease (FMD) is a highly transmissible disease caused by Aphthovirus of the family Picornaviridae. This study aimed to investigate the serological approach (non-structural protein [NSP] analysis) of 3ABC enzyme-linked immunosorbent assay (ELISA) to diagnose FMD cattle in vaccinated and unvaccinated animals. A total of 84 serum specimens, including non-vaccinated, single-vaccinated, and multi-vaccinated samples, were collected from four districts in Baghdad Province, Iraq, to evaluate the antibodies to NSP of the FMD virus. The ELISA was used to detect antibodies (NSP) of FMDV in the serum of cattle. The result showed that the seroprevalence was estimated at 34% (29/84) in farm animals. The seroprevalence rates of FMD in relation to the age of infected animals were obtained at 21%, 7%, and 6% in 9-23-, 24-36-, and ≥,36-month-old groups, respectively. The consequences of the examination of the sera from naive, immunized, and nonimmunized infected farm animals applying 3ABC-ELISA were presented,accordingly, the incidence rates of FMD infection in non-vaccinated and vaccinated animals were 18 (75%) and 11 (18%) respectively. Negative results were recorded in the immunized group 49 (82%) higher than in the non-immunized group 6 (25%). Evaluation of NSP antibodies to isolate vaccinated animals from infected ones showed that the application of these assays was significantly useful for FMD prevention and control management programs in infected areas.