A total of 72 peptonated water samples suspected of carrying Vibrio cholerae were assessed by laser flow cytometry (LFC) and compared with positive culture. We used a direct fluorescence technique using polyclonal (PolAb) and monoclonal antibodies (MoAb) conjugated to fluorescein. The PolAb were able to detect 33 positive samples. A clear difference among the 20 positive samples was found with only three V. cholerae O1 false negatives when MoAb were used whereas all 13 V. cholerae Non O1 samples were detected. The correlation index comparing control autofluorescence with peptonated water samples show a R = 0.69, versus 0.96 with pure V. cholerae O1 strains. Our data suggest that the LFC technique is able to recognize V. cholerae O1 from a mixture of microorganisms with high sensitivity and specificity in a few hours.