The recognition of the viable but non-culturable (VBNC) state of pathogenic bacteria has brought with it many questions to answer related to the need to detect and quantify viable bacteria in the environment in an accurate way. To assess viability of Vibrio cholerae， we developed a RT-Real Time PCR technique based on differential expression analysis from mRNA deep sequencing data. We compared two induction conditions to achieve the VBNC state: a bacterial suspension induced by artificial seawater at 4°C， and the addition of 3'，5'-cyclic diguanylic acid. The evaluation of the up-regulated genes in the induced bacterial samples was compared with a fresh culture in the mid-exponential phase. The data analysis was performed with the NOISeq R-package and revealed 17 up-regulated genes for induction condition I and 22 up-regulated genes for induction condition II. Only one region was found to be up-regulated for both induction conditions. The VCA0656 gene related to the aminoimidazole riboside kinase protein was detected as the most up-regulated region and used as a genetic marker to precisely detect the VBNC state in combination with the RT-Real Time PCR technique. This approach describes a novel method to differentiate the VBNC state of V. cholerae in water samples.