Foot and mouth disease (FMD) is the most important contagious viral disease affect all cloven hoofed animals, causes an economic loss. The present study aimed for studying serological pervasiveness of Foot and mouth disease virus (FMDV) in Qualubeyia Governorate during 2013-2014. It was carried out on 400 serum samples. Antibodies against FMDV nonstructural protein using FMD Blocking ELISA in cattle and buffalo were 70 (35%), 60 (30%) out of 200 serum samples for each species. The Over all positive percent of FMDV antibodies in sera of cattle and buffaloes were higher by ELISA than by SNT. Direct detection of FMDV serotypes in tongue epithelial samples using ELISA showed positive result for FMDV serotype O in 3 out of 40 samples (7.5%). Trials for isolation of FMDV from ELISA positive samples on BHK-21 cell showed positive results with one out of 3 samples that was confirmed as FMDV serotype O using RT-PCR. Finally, it was concluded that antibodies against serotypes O, A and SAT2 FMDV was prevalent in both infected and vaccinated animals using different types of ELISA kits that served as the most suitable, rapid and sensitive techniques. Also, antigen detection ELISA was rapid and more reliable than isolation followed by virus detection for direct diagnosis of different serotypes of FMDV in samples