Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. The LAMP assay requires 12-18 min for amplification with a single colony on selective agar from cholera toxin (CT)-producing Vibrio cholerae strains and less than 60 min with human feces and seafood samples. The assay requires less than 35 and 80 min for the detection of CT-producing V. cholerae with a colony on selective agar and with human feces and seafood samples from the beginning of DNA extraction to final determination. The LAMP amplification can be judged by both turbidimetric analysis and visual assessment with the unaided eye. The sensitivity of the LAMP assay is tenfold higher than that of the PCR assay. The LAMP assay is a powerful tool for rapid, simple, and sensitive detection of CT-producing V. cholerae which may facilitate the investigation of V. cholerae contamination in seafood, as well as the early diagnosis of cholera in humans.