Foot and mouth disease (FMD) is still endemic in Egypt despite regular control program to eradicate. In this study, seroprevalence to determine the distribution of antibodies against FMD virus serotype O and A and differentiation of antibodies produced against naturally infected from vaccinated cattle and buffalo at Mounofya governorate were done. Also direct serotype detection from field samples in the herd was carried out using indirect sandwich ELISA. Results showed that, the antibodies prevalence against serotype A were higher than that against serotype O in cattle and buffalo as detected by Liquid phase blocking ELISA (LPBE) and Serum neutralization test (SNT). Commercial ELISA kit (Priocheck) based on 3ABC non-structural proteins to differentiate infected from vaccinated animal were positive for 35.8% of cattle and 26.0% of buffalo. From a total of 73 samples submitted for direct detection and serotyping of FMDV by indirect sandwich ELISA, 14 were typed as serotype A and 7 as serotype O. Surprisingly both FMDV serotypes O and A were detected in two samples from examined buffy coat. All indirect ELISA negative samples also tested negative for FMDV isolation on BHK-21 cell line. In conclusion, serotype A was the most prevalent serotype in cattle and buffalo at Mounofya governorate, and different ELISA formats were sensitive and rapid techniques for seroprevalance and serotype detection of FMD virus