Abstract: To establish a rapid and efficient serological detection method for cucumber mosaic virus (CMV), CMV antiserum was prepared using prokaryotic expression of the coat protein (CP). The results showed that the gene sequence amplified from infected tobacco had over 95% identity of the nucleotide sequence with those of CMV CP genes, belonging to subgroup IB and possessing structural molecular activity. After IPTG induction of the prokaryotic expression vector pET-28a harboring the CMV CP gene, the CMV CP fusion protein was expressed. Healthy New Zealand white rabbits were immunized with the purified CMV CP fusion protein, and CMV antiserum was successfully prepared. The prepared CMV antiserum exhibited a titer of 1:40 000 and was specific to CMV. CMV antiserum was used to detect suspected field samples by indirect ELISA, and the detection rate of CMV reached 85.2%, demonstrating 92.0% concordance with RT-PCR detection results. The results indicated that the CMV antiserum was suitable for the detection of field samples, laying the foundation for rapid detection and prevention of CMV.