[Objective] To establish a duplex PCR assay for detection of the strains XSBZ03 and XSBZ14. [Methods] Strain-specific primers were designed according to the target sequence of XSBZ03 and XSBZ14. Subsequently, duplex PCR assay based on the primers was successfully established. [Results] The duplex PCR assay could detect accurately strains XSBZ03 and XSBZ14. The detection limit for XSBZ03 and XSBZ14 genomic DNA concentration was 1.7 pg/μL and 2.0 pg/μL. In artificial seawater samples, the detection limit for XSBZ03 and XSBZ14 strain concentration was 6×10³ CFU/mL and 8×10³ CFU/mL, respectively. [Conclusion] This assay provides a reliable method for the rapid diagnosis of Porites andrewsi White Syndrome (PAWS) and the Specific Pathogen Free (SPF) coral transplantation caused by these two pathogens.