A non-radioactive digoxigenin cDNA probe for detection of avian encephalomyelitis virus (AEV) was developed. The cDNA probe hybridized specifically with a fragment of VP1 gene of AEV genome was found to be sensitive with as little as 10 pg target DNA fragment in a sensitivity test. Infected chicken embryos were strongly labelled by testing the probe against a range of AEV strains, and no non-specific reaction was observed in non-infected chicken embryos as well as five other avian pathogenic virus-infected samples used as negative controls. Furthermore, the cDNA probe was capable of detecting AEV from chicken embryo brain at 3 days post-inoculation as compared with an immunofluorescence assay, which required up to 5 days of incubation in the embryos. In clinical application, five out of 16 clinical brain samples that were negative by the immunofluorescence assay were positive for AEV by the cDNA probe. Both the sensitivity and specificity of the developed cDNA probe indicated that it is a highly promising and reliable diagnostic tool for the detection of AEV infections.