In this study, a nested PCR for detecting soft-shelled turtle iridovirus (STIV), wo pairs of primers were designed based on the sequence of major capsid proteins (MCP) gene of STIV by The outer primers were P1 (5′-′CGCGATAGGCTACTAT AACATGG). The first round PCR was carried out using PCR Supermix with 40 ng template DNA, 0.6 µL primers (P1/P2, 50 pmol/µL), 2 µL PCR buffer with MgCl₂, 0.5 µL Tag, and dH₂O was added to reach total volum of 20 µL. The conditions of PCR were: 94 PCR was carried out by using the same supermix except 0.5 µL template DNA, the product of first round PCR, 0.6 µL primers (P3/P4, 50 pmol/µL). The conditions were the same with the first round PCR. In this study, a standard recombinant plasmid was produced to in PCR assay. The DNA products of first round PCR were extracted, and then cloned into pGem-T Easy vector. After sequencing, the -S were used in ranavirus detection as the position control. Specificity and sensitivity of the PCR method were estimated. Data showed that the detection limit of this assay was 10² copies for standard recombinant plasmidIt was also found that the specificity of this assay was high without any cross-reactions with DNA from Infectious haematopoietic necrosis (IHNV), Spring viraemia of carp virus (SVCV), Viral Haemorrhagic Septicaemia virus (VHSV), Channel catfish virus (CCV), Infectious pancreatic necrosis (IPNV), Red sea bream iridovirus (RSIV), Hirame rhabdovirus (HRV), Koi Herpesvirus (KHV) and Infectious spleen and kidney necrosis virus (ISKNV) provided bySTIV, EHNV and TFV DNA could result in the same pattern of curves with the two primers. confirmed that the PCR assay is a powerful tool for the detection of ranavirus with high sensitivity and specificity. Moreover, the is of low cost and time saving in contrast to ELISA assay, . So the Nest-PCR method could be used to detect these three kinds of ranavirus efficiently.