Bluetongue, an economically important arthropod-borne viral disease of livestock especially sheep. Presently, there are 27 serotypes of bluetongue virus prevalent worldwide. Traditional bluetongue virus typing methods such as virus isolation and Serum Neutralization Tests (SNT) are cumbersome, time-consuming, and many times inconclusive. The other diagnostic methods such as antigen-capture enzyme-linked immunosorbent assay (ELISA), dot immunobinding assay and immuno-electron microscopy, have been developed. All these techniques have limited sensitivities and thus are unable to detect the virus at a sub-clinical level. Serotype specific RT-PCR and qRT-PCR based amplification of BTV Seg-2 are most reliable, less time consuming and high specificity for serotype typing. In the present study, standardization of EVA-green based qRT-PCR was done for amplification and quantification of BTV-23 serotype from infected BHK-21 cell culture; the results showed high sensitivity as well specificity of realtime PCR for diagnosis of BTV-23.