Abstract: Vibrio alginolyticus is a major pathogenic bacterium that severely infects farmed marine aquatic animals, including fish, shrimp and shellfish. To achieve rapid and accurate detection of this pathogen, a TaqMan probe-based quantitative PCR (qPCR) assay was developed in this study. Specific primers and a TaqMan probe were designed targeting the conserved region of the vopS gene of V. alginolyticus. The reaction system and amplification conditions were optimized, and the specificity, sensitivity and reproducibility of the assay were evaluated using a plasmid standard curve. The results showed that the assay is highly specific for V. alginolyticus with no cross-reactivity with other aquatic pathogenic bacteria. The limit of detection was 1 copy·μL⁻¹. The standard curve showed a correlation coefficient (r²) of 0.999 2 and an amplification efficiency of 105%. Both intra-assay and inter-assay coefficients of variation (CVs) were below 5%, indicating good stability and reproducibility. Furthermore, the assay successfully detected V. alginolyticus aquaculture water and oyster tissue samples. In conclusion, the TaqMan qPCR assay established in this study is specific, sensitive, and reproducible, and provides a reliable tool for the rapid and accurate detection of V. alginolyticus.