Three different procedures of purification were performed to purify a local isolate Of Barley stripe mosaic Hordevirus (BSMV). The 1st procedure was successful in producing relatively high yield of virus preparation 3.7 mg/100 g of barley leaves with adequate purity and one light-scattering zone was round in sucrose density-gradient column. The 2nd and 3rd procedures yielded aggregated virions (2.3 and 7.0 mg/ 100 g of fresh barley leaves, respectively). Electron microscopy of negatively stained purified virus preparation showed rod-shaped particles with dimension of 150x25 nm. The polyclonal antibodies raised against the local isolate of BSMV had a specific titer of 1:2000. Positive reactions were obtained when purified IgG and IgG conjugate with alkaline phosphatase was 1:1000. The prepared antiserum was used detection of BSMV by serological methods i.e., Enzyme-linked immunosorbent assay (ELISA), Dot-blot and tissues-blot immunobinding assays (DBIA and TBIA). The presence of the virus was confirmed in mature seed parts and non-seed parts of the different five barley cultivars tested.