Objective To establish a new accurate quantitative method for detection of Vibrio alginolyticus by droplet digital polymerase chain reaction PCR (ddPCR) in aquatic products. Methods The primer and probe of Vibrio alginolyticus in Method for the Detection of Pathogens in Food for Export—Real-time PCR Method (SN/T 1870-2016) were selected, and the ddPCR reaction system was established and optimized. The limit of detection(LOD) and the limit of quantitation (LQD) were tested based on ddPCR using pure bacterial genomic DNA and quantitative accuracy analysis was performed on the result. In order to verify the feasibility of the method, positive strain was added to pre-screening squid rings without background and the result obtained by ddPCR and quantitative real time polymerase chain reaction(qPCR)were compared. Results Vibrio alginolyticus performed good specificity in ddPCR detection in this experiment. In pure culture of Vibrio alginolyticus, the LOD and LOQ values overlapped each other, which was 2.2 copies/reaction, corresponding to 110 copies/ml. In the artificial contaminated experiment, the result of ddPCR for quantification of Vibrio alginolyticus were closer to theoretical value, and the repeatability and the detection rate of low concentration sample were also better than qPCR. The LOQ of artificially contaminated sample could reach 120 copies/g. Conclusion The result showed that quantitative detection of Vibrio alginolyticus by ddPCR had good specificity, high sensitivity and accuracy, which had practical application value.