Abstract: Lack of reliable and rapid method for detection of pathogens from shrimp in Bangladesh is an obstacle in ensuring the microbiological quality of the shrimp before export. Salmonella sp. and Vibrio parahaemolyticus are two potential pathogen for shrimp export value chain in Bangladesh. The objective of this study was to establish a multiplex polymerase chain reaction method for rapid and simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus from export oriented shrimp samples. The targeted genes were tdh for V. parahaemolyticus and sefA for Salmonella spp. The genomic DNA was extracted and amplified for subsequent profiling. Validity of the multiplex PCR assay was tested by artificially inoculating the shrimp homogenate with viable cells of target pathogens. The genes were successfully amplified individually and simultaneously both from natural and spiked samples. Sensitivity of the assay was determined to be as low as 104 CFU mL-1. Amplification of DNA extracted from other bacterial pathogens viz. Bacillus cereus, Shigella flexneri and Staphylococcus aureus yielded negative results. This multiplex PCR assay will provide specific, rapid and reliable results and allow for the cost effective detection of target pathogens in a single reaction tube in mixed bacterial communities that are prevalent in shrimp products.