Objective To identify and conduct antimicrobial susceptibility testing on Vibrio species isolated from blood culture specimens of two patients with bloodstream infection (BSI), analyze the microbiological characteristics of non-O1/non-O139 Vibrio cholerae (NOVC), and provide evidence for the diagnosis, prevention and control of Vibrio cholerae infection. Methods Two Vibrio strains were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API bacterial biochemical reaction identification test strip, VITEK 2 Compact identification instrument and 16S rRNA gene sequencing. Serological typing, virulence gene molecular detection, and drug resistance phenotype detection were performed to the Vibrio strains. Results Two strains were identified as Vibrio cholerae, classified as NOVC by serological typing, and were negative for ctxAB virulence gene detection. Antimicrobial susceptibility testing showed that one strain was sensitive to ampici-llin, azi-thromycin, doxycycline and chloramphenicol, while resistant to tetracycline and trimethoprim-sulfametho-xazole. The other strain was sensitive to all tested antimicrobial agents. Conclusion BSI caused by NOVC is rarely reported in China. Accurate identification, typing and drug-resistant phenotype detection of Vibrio cholerae isolated from blood culture specimens are valuable for the diagnosis, treatment, prevention and control of Vibrio cholerae-associated infection.