This work aimsto prepare and evaluate Rift Valley Fiver (RVF) antigen for detection of RVF antibodies in infected and vaccinated animals. Three types of antigen (whole cells suspension , cell lysate and supernatant) were prepared and to be proved sterile and safe when inoculated in tissue culture and mice, and revealed positive reaction in AGPT with specific reference antiserum and checkerboard ELISA was used for titration of the prepared antigens. The obtained results revealed that the whole cell antigen had a titer of 1/160, and the cell lysate antigen had a titer 1/200 while the supernatant antigen was of a titer 1/20. Application of these antigens with the serum samples obtained from vaccinated animals with RVF inactivated vaccine, revealed that the cell lysate antigen is more potent followed by the whole cell suspension one for detection of anti RVF IgG than other supernatant antigen. From the previous results, it can be concluded that the cell lysate antigen is the best and can be used to detect the anti-bodies of the RVF in the animal vaccines.