Foot-and-mouth disease (FMD) is a highly contagious and economically important disease of cloven footed domesticated and wild animals. Lot of works have been carried out to develop diagnostic tests in regard to this disease The present study was undertaken for detection of FMDV serotype ‘O’ in infected as well as in recovered animals by using sandwich ELISA, virus isolation in cell culture in BHK-21 cell line and reverse transcription PCR and the finding were compiled together to see the most sensitive test for detection of FMDV type ‘O’. A total of 50 clinical samples comprising 40 tongue epithelium, 6 oral swab and 4 throat swab samples were subjected to sandwich ELISA where 20 were found positive for FMDV type ‘O’ (40%),  where as all the swab samples were found to be negative for any serotype of FMDV. Sandwich ELISA positive samples and all the swab samples were subjected to virus isolation where only 11 samples showed CPE (36.66%). FMD virus serotype ‘O’ could be detected in all samples (100%), by RT-PCR using serotype ‘O’ specific primer. Therefore, the present study revealed that RT-PCR is most sensitive for the detection of RNA of FMD virus when compared to the results of the three tests.