Abstract An outbreak of suspected iridovirus disease in cultured hybrid grouper (♀Tiger Grouper Epinephelus fuscoguttatus × ♂ Giant Grouper Epinephelus lanceolatus) occurred in the Guangxi Province in July, 2018. In this study, grouper iridovirus Guangxi (SGIV-Gx) was isolated from diseased hybrid grouper that were collected from Guangxi. Cytopathic effects were observed and identified in grouper spleen cells that were incubated with diseased tissue homogenates after 24 h, and the effects increased at 48 h postinfection. The transmission electron microscopy results showed that viral particles that were about 200 nm in diameter with hexagonal profiles were present in the cell cytoplasm of suspected virus-infected cells. The presence of SGIV-Gx (accession number: MK107821) was identified by polymerase chain reaction (PCR) and amplicon sequencing, which showed that this strain was most closely related to Singapore grouper iridovirus (AY521625.1). The detection of SGIV-Gx infection was further supported by novel aptamer (Q2c)-based detection technology. The effects of temperature and pH on viral infectivity were analyzed by using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and cell culture. The results indicated that SGIV-Gx was resistant to exposure to pH levels 5, 7, and 7.5 for 1 h, but its infectivity was remarkably lower at pH levels 3 and 10 after 1 h. The analyses showed that SGIV-Gx was stable for 1 h at 4°C and 25°C but was inactivated after 1 h at 40, 50, and 60°C.