In order to find out the prevalence status of cow brucellosis in Yongjing County of Gansu Province and to determine the types of infection strain，during 2013 to 2017，brucellosis surveillance was carried out towards cows over 8 months of age，and 3 spleen samples were detected Brucella-positive in 2016. Then isolation and identification of Brucella in suspected samples was carried out. Results showed that during 2013 to 2014，444 cows were tested，no positive cow was found；during 2015 to 2016，706 cows were tested，and 63 of them were detected brucellosis-positive，the individual positive rate was 8.92%. For the 3 suspected spleen samples，PCR tests all showed positive results. After 15 days of bacteria isolation and culture，bacteria colonies only grew in one culture plate. At last，one bacterial strain was isolated. Through AMOS-PCR detection，the amplified band revealed the characteristic of Brucella abortus，also，the sequencing results of omp25 gene of the isolate showed high consistency with that of Brucella abortus. In conclusion，the research results would provide technical supports for brucellosis prevention and control in Yongjing County.