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Abstract Brucellosis remains one of the most common zoonoses spread worldwide, inducing enormous economic losses to the livestock industry and posing serious health threats to humans. Brucellosis re-emerged in China in the mid-1990s and reached a historically high level in 2015. The National Brucellosis Prevention and Control Plan (NBPCP) was initiated from 2016 to 2020. However, the present epidemiological status in livestock has not been elucidated, and whether Brucella variation occurred remains unclear. This study performed an extensive serological investigation in ruminant livestock from 2019 to 2021 in central Gansu Province, China. In total, 11,296 samples from 337 farms were collected to detect the specific antibodies of Brucella. The yearly average serological prevalence of Brucella at the flock level and individual level declined from 11.32% to 8.26% and 1.17% to 0.57%, respectively. The apparent individual-level seroprevalence of small and large ruminants was 0.89% and 0.52%, respectively. The brucellosis distribution has shifted from pastoral areas to agro-pastoral areas. Flock size and gender may be major risks of Brucella infection. Then, the B. melitensis TZ strain was isolated from female Tibetan sheep blood cell lysates. Phonotypical characterization demonstrated that it belongs to B. melitensis. biovar 3, and multilocus sequencing typing results indicated that it belongs to ST8. The whole genome and subsequent phylogenetic analysis demonstrated that the B. melitensis TZ strain is genetically more closely related to the B. melitensis QH61 strain. The B. melitensis TZ strain has similar growth characteristics to the B. melitensis 16 M strain. Overall, our study suggests that after strengthening control and prevention measures based on the NBPCP, there is a very low prevalence or absence of B. melitensis in the central Gansu Province of China, and the genotype of an epidemic strain of Brucella in Northwest China is relatively stable.