In this study, we developed a multiplex reverse transcription-polymerase chain reaction for simultaneous detection of six major shrimp viruses including white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvo virus (HPV), Taura syndrome virus (TSV), Baculovirus penaei (BP) and infectious myonecrosis virus (IMNV). The reaction condition was optimized, and the specificity and sensitivity was tested for this method. In 50 µl reaction system, the optimum concentration of Mg²⁺ was 5mmol/L, optimum dosage of ExTaq enzyme was 3.75U, reaction procedure in the optimal annealing temperature was 55.5℃. Between six kinds of virus and shrimp genome exist good specificity. The detection limits were 104copies for the detection of WSSV, 10² copies for IHHNV, 10⁴ copies for HPV, 10 ³ copies for TSV, 10⁵ copies for BP, and 10⁵ copies for IMNV. Although the sensitivity is not as good as a single PCR,the actual sample testing validated that the method time-saving and less reagent consuming.