Vibrio cholerae is the etiological agent of epidemic cholera. Symptoms include stomach ache, bloody diarrhea, and vomiting, which lead to severe dehydration and even death. Environmental monitoring as well as rapid and accurate identification of this pathogen are important for public health protection. In this study, a real-time PCR method for the detection of toxigenic V. cholerae was developed. In total, 63 environmental and clinical strains were tested for the presence of seven targets, namely ompW, ompU, tcpA, ctxA, zot, rfbO1, and rfbO139. The proposed method is specific, simple, and fast, and can be used for detection of toxigenic and non-toxigenic V. cholerae strains. The minimum detection limit of this assay for V. cholerae in environmental water samples was 1.4 CFU/ml.