[Objective] The SD-EMA-PCR was established by incorporating sodium deoxycholate (SD) into EMA-PCR assay for discrimination viable and dead cells of Vibrio parahaemolyticus. [Methods] The optimal concentration of the sodium deoxycholate, the concentration of ethidium bromide monoazide (EMA) for discrimination viable and dead cells, the optimal light exposure time for acativating and photolyzing EMA were determined respectively. And the detection limit value of V. parahaemolyticus in the mixtures of viable and dead cells was obtained by SD-EMA-PCR. [Results] The results showed that when the concentration of sodium deoxycholate is less than or equal to 0.5 g/L, the concentration of EMA is ranged from 3.2 to 34.0 mg/L, the light exposure time is 25 min, the DNA amplification from the dead cells by SD-EMA-PCR were inhibited. The detection limit of the SD-EMA-PCR assay for the viable cells was 10 CFU/mL. [Conclusion] SD-EMA-PCR can be used to minimize the false-positive results by inhibiting the EMA-PCR amplification of V. parahaemolyticus dead cells from a mixed bacterial population. It is an efficient new approach for distinction between the viable and dead cells and efficiently avoid false positive results.