Abstract: The DNA of bovine leukemia virus (BLV) provirus was extracted from fetal lamb kidney (FLK) cell line persistently infected by BLV. The env（gp51）gene which encode gp51 protein was amplified by PCR. Sequencing results showed that the amplified env gene was a 828 bp fragment, the gene was inserted into the expression vector pET-32a, the recombinant plasmid was named pET-32a-gp51. A fusion protein was expressed in BL21 (DE3) that transfected by pET-32a-gp51 and induced by IPTG. The molecular weight of the recombinant protein was about 43 000 by SDS-PAGE, and the immunoreaction activity of the recombinant protein was confirmed by Western-blot, in which positive serum against BLV was used. An indirect enzyme-linked immunosorbent assay for detecting antibody against BLV was developed by using expressed protein gp51 as coating antigen. Results showed that the optimal coating concentration for gp51 is 2.19 μg·mL^-1 and the serum dilution fold is 1∶80. The positive criterion for this ELISA assay is OD450 nm> 0.451. Accuracy rate for detecting 12 standard BLV sera is 91.67%.