Abstract: Objective To study on epidemiology and molecular characteristics of Brucella melitensis during an zoonotic disease outbreak of brucellosis in sheep farm. Methods Brucella antibodies were detected by serum agglutination testing (SAT). The disc diffusion method was used for antibiotic susceptibility testing of Brucella melitensis strains. Molecular characteristics was analyzed by multi-variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST). Results The seroprevalence of Brucella antibodies in humans and sheep in sheep farm was 7/18 and 5/8 respectively.Two Brucella melitensis strains were cultured from the blood of two sheep keepers (No. CQ-18 and CQ-11). Three Brucella melitensis strains were cultured from the blood of sheep (No. CQ-154,CQ-159 and CQ-237). Antibiotic susceptibility test results showed that five strains were sensitive to ciprofloxacin,levofloxacin,doxycycline,ceftriaxone,norfloxacin,minocycline,moxifloxacin,ofloxacin,rifampicin and streptomycin. Two strains of CQ-154 and CQ-237 were sensitive to SMZ-TMP. Three strains of CQ-159,CQ-18 and CQ-11 were resistant to SMZ-TMP. All five strains were resistant to azithromycin and clarithromycin. MLVA analysis found that the strain CQ-154 lacked a tandem repeat in the BRU16 locus. The strain CQ-18 had an increased tandem repeat in the BRU04 locus. MLST results showed that all isolated strains could be classified into a 8 ST type. Conclusion It was a key problem for people and livestock can not cure the infection of brucellosis because of Brucella can harbor within animal body for a long time. It was the first time to find that Brucella melitensis was resistant to SMZ-TMP in China.