Abstract: Based on collagenase gene sequences of Vibrio parahaemolyticus and omp W gene sequences of V.cholerae,designed two pairs of DPO( dual priming oligonucleotide) primers. A multiplex DPO-PCR method had been developed for the detection of V. parahaemolyticus and V. cholerae simultaneously. The specificity and sensitivity of the method had been tested. The result showed that the DPO primers were of high specificity.Amplified fragments 307 bp for V.parahaemolyticus and 463 bp for V.cholerae were obtained,the sensitivity of the method was 0.1 ng / μL for V. parahaemolyticus and V. cholerae. The multiplex DPO-PCR was not sensitive to the annealing temperature.The multiplex DPO- PCR was validated with 69 suspicious vibrio strains and the results was consistent with physiological and biochemical experiments. The multiplex DPO-PCR method proved to be strong specificity,high sensitivity.This method was suitable for the detection of V.parahaemolyticus and V.cholera in food,aquatic products,etc.