Objective To establish a dual RT-PCR detection method for bovine viral diarrhea virus (BVDV) in bovine-derived samples. Methods The primers were designed and synthesized according to the published BVDV1 and BVDV2 genes containing highly conservative sequences in the 5' untranslated regions (5' UTR) to establish the dual RT-PCR method. The specificity, sensitivity, stability of this method were evaluated. Then 41 bovine-derived samples and 64 bovine plasma samples including bovine calf serum, deproteinized calf serum extract and one lienal polypeptide injection were detected with this method. Results There was no cross reaction with bovine parainfluenza virus type 3 (BPIV3), classical swine fever virus (CSFV) and Japanese encephalitis virus (JEV) when samples were detected with the established dual RT-PCR method. The lowest concentration of template DNA for detection of BVDV1 and BVDV2 was 8.87×10² copies and 6.31×10² copies per microliter, respectively. Electrophoresis bands of about 151 bp and 303 bp were still amplified and detected after the BVDV1 and BVDV2 cDNA was stored at -30℃ for 12 months. The BVDV positive rate of 41 bovine-derived samples and 64 bovine plasma samples detected with this dual RT-PCR method was 14.6% and 29.7%, respectively. Conclusions The established dual RT-PCR method has the advantages of high efficiency, specificity, sensitivity and stability, and can be used for the detection of BVDV in bovine-derived samples.