Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of sheep and goats, associated with the oncogenic retroviruses enzootic nasal tumor virus (ENTV) 1 and 2, respectively. It appears to be common in countries with substantial small ruminant-production. ENA diagnosis in goats is based on autopsy and histopathology, and there is no real-time PCR method available for ENTV-2 detection. Here, a novel one-tube real-time RT-PCR (RT-qPCR) method for the detection and quantification of ENTV-2 in nasal swabs is presented. The method targets the env gene/U3 region. For the design of ENTV-2-specific oligonucleotides, molecular characterization of seven Greek ENTV-2 strains was performed. Phylogenetic analysis revealed three distinct phylogenetic clades of ENTV-2 that correlate with the country of sample collection. Evaluation of the analytical performance of the RT-qPCR revealed an amplification efficiency of 92.8% and a linear range of quantification between 2 × 108 and 2 × 102 RNA transcripts. Analysis of nasal swabs from 23 histopathologically confirmed, naturally occurring ENA cases via RT-qPCR yielded positive results. Moreover, modification of the method for use in a real-time PCR (qPCR) assay enables detection of proviral DNA in tumor specimens. Both methods are highly specific and can be used for the confirmation of ENA-suspected cases. Future applications could include ante-mortem diagnosis, verification of the ENTV-2-free status in animal trade, disease surveillance, and control programs.