Vibrio alginolyticus is widely distributed in marine ecosystems. V. alginolyticus is also associated with infection in aquatic animals, and has a significant negative impact on aquaculture. Therefore, devising a rapid, sensitive, and effective detection method for V. alginolyticus is necessary. In this study, we developed a detection method for V. alginolyticus using loop-mediated isothermal amplification (LAMP) assay, in which the V. alginolyticus gyrB (DNA gyrase subunit) gene was used as the target gene. A set of primers was designed to amplify specific DNA sequences by LAMP. Moreover, the reaction conditions were optimized. Results show that the optimum conditions for LAMP assay for the rapid detection of V. alginolyticus were 63.5 °C for 45 min. The optimum concentrations of Mg²⁺ and dNTP were 5 and 1 mM, respectively. The LAMP assay had a detection limit of 2.08 × 10^–5 ng/µl. LAMP detection of V. alginolyticus is more simple and rapid compared with traditional detection methods. The results of LAMP detection can be identified without the aid of sophisticated equipment or a complicated protocol. Thus, the LAMP assay is a potential diagnostic tool for V. alginolyticus.