Vibrio harveyi is one of the opportunistic marine environment bacteria that are considered as one of the main causes of Asian seabass vibriosis diseases in many marine cage farms in the world. The aims of this study were isolation and identification of these bacteria in cultured Asian seabass in farms, located southern provinces of IRAN (Khuzestan, Bushehr and Hormozgan). In this study, a total number of 110 Asian seabass (80, with clinical sings of disease and 30, apparently healthy fish) with different body weight ranging 50-700g were sampled. Isolation was done from internal organs (kidney, heart, spleen and liver) in common method of bacterial isolation and isolates subjected to phenotypic and molecular characteristics using duplex PCR amplification of the 16SrDNA & vhh genes respectively genus & species-specific gene. The results of PCR showed that of the 95 phenotypically identified isolates, only 65 isolates belong to the genus vibrio and 46 (70.76%) of them were identified as Vibrio harveyi. These results indicate a high present of Vibrio harveyi in cultured Asian seabass in studied farms. Results of the biochemical characteristics of the isolates confirmed with PCR, showed isolates have different reaction in several biochemical tests. Accordingly, the duplex PCR method presented in this study was introduce as an accurate and rapid method for diagnosis of V. harveyi.