“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 evaluation 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, and it was cloned and further used to construct plasmid standards of qPCR; the cycle threshold value (CT) 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 method could specifically detect AEAdoV with negative amplification reaction on Rana grylio virus (RGV), Anguilla 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%, showing its 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 on the virus quantity of the eel tissues indicated that the level of AEAdoV was relatively higher in the organs of heart, intestine, gill and fin, and lower in mucus and skin and spleen. This study will valuable for furether studying the correlation between AEAdoV and HGND, and helpful for understanding the epidemic and etiology of AEAdoV.