[Objective] Deepen our understanding about the diversity of epidemic arboviruses of Yunnan Province [Methods] Blood samples regularly collected form sentinel cattle placed in Jinghong city of Yunnan Province, were inoculated on cells for arboviruses isolation. The isolated virus strain was initially identified by RT-PCR, viral genomic agarose gel electrophoresis (AGE) and electron microscope (TM). Full-length segment 2 (Seg-2) and segment 3 (Seg-3) of the isolated virus were sequenced and the virus classification was conducted by phylogenetic tree construction. The infection kinetics of the isolated virus on the sentinel cattle was retrospectively traced back by qRT-PCR and serum neutralization test.[Results] An unidentified virus strain, causing significant cytopathic effect on BHK-21 cells, was isolated from the blood sample of sentinel cattle. The genome of isolated virus is composed of double stranded RNA showing "3-3-3" migration pattern by AGE analysis. TM observation revealed that the virus particles exhibited typical characteristics of Orbivirus with ring-shaped structure about 70 nm in diameter. Seg-2 of the isolated virus encoding the conserved T2 protein of Orbivirus, showed the closest relationship with Mobuck virus with nucleic acid (nt) and amino acid (aa) sequence identities of 72.8% and 84.0% respectively. Meanwhile, Seeg-3 of the isolated virus encoding Orbivirus serotype decisive OC1 protein, only showed 60.2% (nt) and 56.5% (aa) sequences identity with Mobuck virus. Furthermore, on the phylogenetic tree of T2 and OC1, the isolated virus formed a distinctive branch from Mobuck virus. Retrospective tracing results of the virus infected cattle showed viral RNA lasted in the blood for 17 weeks until supervision terminated. Neutralization antibodies (antibody titer 1:14) were detected one week after virus infection, peaked at the 9^th week (antibody titer 1:226) and remained at a high level (antibody titer 1:113) to the end of monitoring. [Conclusion] We reported here, for the first time, the isolation and infection kinetics of a novel Mobuck virus strain isolated from sentinel cattle in Yunnan Province. The results of this study enriched our knowledge about orbiviruses in China, and laid a foundation for the diagnosis, epidemiology and pathogenicity of this novel virus.