Vibrio vulnificus is one of the major cause of foodborne illness related to seafood consumption. This study aimed to develop monoclonal antibodies (MAbs) to enable rapid detection and monitoring food contamination of this pathogen. Five groups of MAbs against V. vulnificus were generated from a mouse immunized with a clinical isolate of V. vulnificus, heat and formalin-inactivated whole cells. The first two groups of MAbs were species-specific to V. vulnificus, recognized all 40 isolates of the bacterium without cross-reactivity to other Vibiro and non-Vibrio species, but bound to different protein antigens. The MAbs in the third group recognized four of nine clinical isolates and two isolates from seafood samples and bound to lipopolysaccharide antigens. The MAbs in the fourth group bound only to all isolates of V. vulnificus and Vibrio alginolyticus, and the MAb in the fifth group was genus-specific bound to all isolates of Vibrio species tested. Based on Dot-enzyme-linked immunosorbent assay, the range of detection sensitivity limits of these MAbs were 105 to 107 CFU/ml, which is 10–1,000 times less than that of direct PCR detection (104 CFU/ml). However, after pre-enrichment of an oyster sample in tryptic soy broth for 6 hr, the detection limit was improved to 1 CFU/ml. This detection sensitivity limit is comparable to that of PCR protocols, which mostly require a pre-enrichment step in sample preparation. The sets of MAbs could provide valuable tools for simple, fast, and low-cost detection of V. vulnificus and Vibrio species in seafood, clinical, and environmental samples.