Abstract: “Cracked head disease” is one of the major diseases in cultured yellow catfish（Pelteobagrus fulvidraco）. In this study, two special oligonucleotide primers for duplex PCR amplication were designed to detect the pathogen of“cracked head disease”from yellow catfish（Pelteobagrus fulvidraco）. The reaction conditions of the duplex PCR were optimized and PCR products were sequenced. Specificity and sensitivity of duplex PCR were studied. Finally, the duplex PCR is well developed successfully allowing the detection of the two bacterial pathogens in one PCR tube with relatively equal intensities DAN bands when analyzed in an agarose gel. The result showed the expected DNA fragments of 470 bp and 268 bp were observed, respectively. The sensitivity of duplex PCR was 1.38 ng/μL. When it was applied to detect the bacterial in brain of fish which were naturally infected in Nanchang, Jiangxi Province, Edwardsiella ictaluri was detected in 11 diseased samples of yellow catfish. The result was coherence with that of traditional technology of physiology and biochemistry, which showed that the duplex PCR could be used not only to diagnose the diseased yellow catfish but also to monitor the fish infected by bacteria. It can be concluded that the duplex PCR is specific and sensitive. It is a reliable tool for identification of the Edwardsiellosis with less time and cost, and it can be used in quick diagnose and epidemiology investigation of bacterial of “Cracked head disease” from yellow catfish.