The bovine enterovirus (BEV) is a pathogen found the digestive tracts of cattle. Recently, the BEV was discovered in cattle in a province in China. A rapid and effective detection method for the BEV is essential. An assay was carried out using two specific primers designed to amplify a highly conserved sequence of the 3D gene. A recombinant plasmid containing the target gene 3D was constructed as a standard control. The limit of detection of the reaction was 7.13 x 10(1) plasmid copies/μL of initial templates, which was tenfold more sensitive than the conventional reverse-transcription-polymerase chain reaction (RT-PCR). Moreover, the assay was highly specific because all negative controls and other viruses of clinical relevance did not develop positive results. Assay performance on field samples was evaluated on 44 (41 diarrhea and 3 aerosol) samples and compared with the conventional RT-PCR assay. Sixteen diarrhea samples were positive (16/41, 39. 02%) and 3 aerosol samples were positive (3/3, 100%). Preliminary results for clinical detection showed that the SYBR Green I real-time PCR assay was highly sensitive, specific and reproducible. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications for epidemics and in BEV research.