Vibrio vulnificus is a zoonotic pathogen linked to aquaculture that is spreading due to climate change. The pathogen can be transmitted to humans and animals by ingestion of raw shellfish or seafood feed, respectively. The aim of this work was to design and test a new procedure to detect V. vulnificus hazardous to human and/or animal health in food/feed samples. For this purpose, we combined a pre-enrichment step with multiplex PCR using primers for the species and for human and animal virulence markers. In vitro assays with mixed DNA from different Vibrio species and Vibrio cultures showed that the new protocol was 100 % specific with a detection limit of 10 cfu/mL. The protocol was successfully validated in seafood using artificially contaminated live shrimp and proved useful also in pathogen isolation from animals and their ecosystem. In conclusion, this novel protocol could be applied in health risk studies associated with food/feed consumption, as well as in the routine identification and subtyping of V. vulnificus from environmental or clinical samples.