Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leucosis. Here， we designed a p24 enzyme-linked immunosorbent assay (ELISA) to detect antibodies specific for BLV capsid protein p24 (encoded by the gag gene) in bovine serum samples. The p24 gene was inserted into an Escherichia coli expression system， and recombinant proteins (GST-p24， p24， and His-p24) were purified. His-p24 was the most suitable antigen for using in the ELISA. The cut-off point was calculated from a receiver operating characteristic curve derived from a set of 582 field samples that previously tested positive or negative by BLV-CoCoMo-qPCR-2， which detects BLV provirus. The new p24 ELISA showed almost the same specificity and sensitivity as a commercial gp51 ELISA kit when used to test field serum samples， and allowed monitoring of p24 antibodies in raw milk and whey. Comparing the results for the p24 ELISA and gp51 ELISA revealed that p24 antibodies were detected earlier than gp51 antibodies in three out of eight calves experimentally infected with BLV， indicating improved detection without diminishing BLV serodiagnosis. Thus， the p24 ELISA is a robust and reliable assay for detecting BLV antibodies in serum or milk， making it is a useful tool for large-scale BLV screening.