Foot-and-mouth disease (FMD) is caused by FMD virus (FMDV) is a highly contagious disease of ruminants, which is primarily controlled by vaccination. The monitoring of antisera after vaccination is currently depending on liquid-phase blocking ELISA (LPBE). Recently, bacterium-original FMD virus-like particle (VLP) showed the potential as vaccine candidates. In this study, to minimize the risk of live virus involvement, the original VLP of FMDV serotype O were used as the immunogen for monoclonal antibodies (Mabs) production and the capture antigen in the development of a solid-phase competition ELISA (SPCE). The samples with a percentage inhibition of >50% were considered positive in the SPCE assay. The concordance rate of the Mab-based SPCE compared with the LPBE for clinical serum samples test was 93.4%, and with a high agreement (kappa = 0.892) with LPBE in antibody duration monitoring. Results indicated that the VLP-based SPCE had high specificity and sensitivity, which provides an alternative method for postimmunization antibody evaluation of FMDV serotype O.