Objective In order to provide scientific basis for Brucellosis control and prevention, sero-epidemiological survey, Brucella isolation and genetic characteristic analysis were conducted for the high-risk population in a human Brucellosis epidemic in Weining county of Guizhou province. Methods Tube agglutination test was used to detect the anti-Brucella antibody for the high-risk human population. Rose Bengal Plate Test was applied to detect the antibody in goat blood samples. The blood of antibody positive human population was collected for Brucella isolation. Conventional methods, genus specific BCSP31-PCR and species-specific AMOS-PCR were used to identify the bacteria strain, and the genetic characteristic was analyzed using MLVA and MLST techniques. Results Six people of 43 high-risk populations were confirmed as anti-Brucella antibody positive, 64 out of 302 goat blood samples were anti-Brucella antibody positive, with positive rate of 21.19%. A suspected Brucella strain were isolated from one of the high-risk human populations and was identified as B. melitensis biovar 3. MLVA-16 analysis indicated that the bacteria strain was most closely clustered with B. melitensis biovar 3. Multilocus sequence typing analysis indicated the strain was ST8. Conclusion Genetic characteristic of Brucella strain isolated from the Brucelosis epidemic was consistent with that of M. melitensis biovar 3. Although antibody and Brucella were detected, the high-risk populations did not displayed symptoms, so all of them were asymptomatic infections. The epidemic was seemingly imported due to goats trading, suggested that health and animal disease control and prevention departments and doctors should pay great attention to it.