Haemorrhagic gill necrosis disease (HGND) has become one of the important epidemic diseases of cultured American eels. In previous studies, American eel adomavirus (AEAdoV) was isolated from eel with HGND. In order to establish a PCR and real-time fluorescence quantitative PCR (qPCR) method for the detection of the virus, primers were designed according to the superfamily 3 helicases (S3H) sequence of AEAdoV-FJ. Further evaluations on the sensitivity, specificity, repeatability, and application effect of this method were conducted. The results showed that the established PCR method could specially amplify a 300 bp band, which was cloned and used to construct plasmid standards for qPCR; the cycle threshold value (C_t) of qPCR and the copy number of the standard sample had a consistent relationship with a wide range. The obtained correlation coefficient (R²) of the standard curve reached 0.999, and the amplification efficiency was 105.067%. The qPCR method could detect a minimum of 10 viral copies with higher sensitivity than routine PCR method. Both methods could specifically detect AEAdoV with negative amplification reaction on Rana grylio virus (RGV), Anguillid herpesvirus (AngHV), Koi herpesvirus (KHV), White spot syndrome virus (WSSV), Japanese eel adomavirus (JEAdoV), and Marbled eel adomavirus (MEAdoV). The coefficient of variation within and between groups of this method were both less than 2%, suggesting good repeatability. Further application results showed that the detection rate of AEAdoV from 35 samples of American eels with HGND by routine PCR and qPCR was 82.8% and 97%, respectively, indicating that the virus is prevalent among the diseased eels. Analysis of the virus quantity of the eel tissues indicated that the level of AEAdoV was relatively higher in heart, liver, gill and fin, and lower in mucus, skin and spleen. These results indicate that routine PCR and qPCR assays with high sensitivity and specificity for detection of AEAdoV have been established, and confirm that AEAdoV is closely related to HGND and exists in the main organs of infected eels. This study will be valuable for further investigations of the correlation between AEAdoV and HGND, and helpful for understanding the epidemic and etiology of AEAdoV.