Abstract: A SYBR Green Ⅰ RT-qPCR assay was established for detecting the pathogenic Mycoplasma ovipneumoniae in sheep.Using Beacon Designer 7.9 software, a pair of specific primers based on the P80 gene of the M.ovipneumoniae strain Y-98 in Genbank (accession number. No.KM435069.1) was designed for the experiment. The result showed that the new methodology could specifically detect the target pathogen but not any others from sheep or goats. The detection limit was 10 copies·μL^-1, and the Ct of intra-and inter-variation all below 2%. The assay method was subsequently applied on 96 clinical samples to yield a positive rate of 67.7% (i.e., 65 out of 96), which was as sensitive as TaqMan RT-PCR. It suggested that the newly established methodology was high on sensitivity, specificity and reproducibility, and applicable for the diagnosis and epidemiologic studies on M.ovipneumoniae.