Background and Objectives: In recent years, the prevalence of diseases caused by Vibrio spp. is increasing in the world, and among them species, Vibrio cholerae is the most important Vibrio associated with pandemic and epidemic cholera outbreaks. Therefore, the development of a reliable method for early and accurate detection of V. cholerae for management of diseases is a real need. Aptamers with the ability to detect targets with high specificity and accuracy can be one of the candidates used for the whole cell and thereby V. cholerae detection. Materials and Methods: In this research high-affinity DNA aptamers against with two major serotypes of Inaba (ATCC 39315) and Ogawa (clinical sample) were selected from DNA aptamer library through 12 rounds of Systematic Evolution of Ligands by Exponential (SELEX) enrichment procedure using live cells as a target which monitored with flow cytometry. Results: The binding efficiency and dissociation constant of the isolated aptamers V.ch47 and V.ch27 were 56.4%, 53.3% and 15.404 ± 4.776 pM, 20.186 ± 3.655 pM, respectively. A sandwich Enzyme-linked aptamer sorbent assay (ELASA) was developed with the biotinylated V.ch47 aptamer and our previously developed nanobody anti-Lipopolysaccharides (LPS). We optimized this system with V. cholerae O1 and analyzed their cross reactivity with close physiological bacteria. The threshold of detection was obtained 104 CFU/ml in the sandwich ELASA process. Conclusion: Our results showed that the sandwich ELASA is sensitive enough for the rapid detection of V. cholerae from other bacteria.