Vibrio is a bacterial genus widely distributed in natural aquatic systems. Some Vibrio species are pathogenic and can cause severe diseases in both marine organisms and humans. Previous studies revealed a link between the current climate change and increased incidence of the Vibrio-associated diseases recently causing sanitary, economic and/or ecological problems worldwide. The conventional culture-based methods (e.g. cultivation of TCBS agar) used to monitor the presence of Vibrio spp. in environmental samples are not always straightforward and can underestimate the number of cells, especially if the microbial population contains a fraction of ‘dormant’ cells (e.g. cells in Viable but not Culturable (VBNC) state). However, this problem can be overcome by using alternative culture-free approaches such as Catalyzed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH). To optimize CARD-FISH for efficient and reliable detection of Vibrio spp. in environmental samples, we have used both computer-assisted and experimental approaches. Our results demonstrate that the use of the optimized protocol along with a very specific probe, ViB572a, can offer the high sensitivity and selectivity of CARD-FISH detection of marine vibrios in natural seawater.