Abstract: Perkinsus sp and Marteilia refringens are responsible for significant economic loss in the shellfish industry. In order to identify Perkinsus sp and Marteilia refringens simultaneously and massively, two pair of primers and two TaqMan probes were designed and synthesized according to the conserved gene sequences of Perkinsus sp and Marteilia refringens in GenBank. The reaction parameters such as the concentration of two pair of primers, two TaqMan probes and the reaction buffer were optimized to develop a duplex real-time PCR assay for the rapid detection of Perkinsus sp and Marteilia refringens. The duplex real-time PCR assay was found to be specific and able to detect and differentiate Perkinsus sp and Marteilia refringens, and no positive results were observed when nucleic acid from Haplosporidium sp, Aeromonas hydrophila, Pseudomonas fluorescens, Vibrio parahaemolyticu, Vibrio Alginolyticu, Vibrio Fluvialis and Vibrio Mimicus were used as duplex real-time PCR templates. The sensitivity of the developed duplex real-time PCR assay was 40 template copies for Perkinsus sp and Marteilia refringens. The samples were examined using the duplex real-time PCR repeatedly and the results indicated that the duplex real-time PCR was reproducible. When different concentrations of Perkinsus sp and Marteilia refringens mixed together still could be identified by this assay, which implied the assay could be applied to clinical confirmation for simultaneous infection of Perkinsus sp and Marteilia refringens. The duplex real-time PCR results of the samples showed that one specific amplified curve was displayed when shellfish was infected by only one of these two protozoan pathogens, whereas two specific amplified curves were displayed when shellfish was infected by two protozoan pathogens. The result indicated that duiplex real-time PCR was able to detect and differentiate the presence of each protozoan pathogen in infected clinical shellfish. This duplex real-time PCR assay was a quick, sensitive, specific and quantitative tool for detection of protozoan, and will be useful for the control of protozoan parasites in shellfish.