In this study, we developed a novel method for the detection of Vibrio alginolyticus, utilizing the specific recognition of an aptamer for V. alginolyticus and signal amplification via hybridization chain reaction (HCR) and horseradish-peroxidase-conjugated streptavidin. The proposed HCR-based multivalent aptamer (multi-Apt) amplifier allows for sensitive detection of V. alginolyticus in a linear range from 10 to 107 CFU/mL. The linear equation is y = 747.5x + 126.2, R2 = 0.986, and the limit of detection (LOD) is 3 CFU/mL. Seawater and freshwater samples were utilized in the spike recovery experiment, yieldng a recovery rates ranging from 94.3% to 108.8%. The relative standard deviation (RSD) for all samples is below 6.73%. Taken together, the proposed method has great potential for application in monitoring of V. alginolyticus in aquaculture environments.