Objective To identify a sporadic case of brucellosis found in Shanghai by bacteriological and molecular biological methods. Methods Blood culture combined with automatic biochemical identification system was used to isolate the Brucella strain from a patient suspected of brucellosis and a Brucella's specific secretary gene was amplified from the strain and analyzed by sequencing. We firstly multiple loci VNTR (Variable Number of Tandem Repeats) was used to analyze the Brucella genotype, by which Brucella dendrogram tree was established based on VNTR fingerprints according to the international published VNTR genotypes data. Results The isolated strain from this sporadic case was identified by bacteriological and molecular biological method as a kind of Brucella species. Although biochemical analysis and special gene sequencing could not identify the biovar of Brucella species, VNTR dendrogram of clustered VNTR genotypes indicated the case of brucellosis belonged to Brucella suis biovar 2.Conclusion Conventional bacterial cultures with biochemical and Brucella VNTR analysis can be used to identify the infection of Brucella and it is important investigate the molecular epidemiological profile of strains isolated in our country.