Early and precise identification of illnesses linked to the bovine leukemia virus (BLV) is crucial for maintaining food safety and efficient herd management in nations like Japan, where milk and beef are staples of diets. Enzootic bovine leukosis (EBL), a B-cell lymphoma caused by BLV, emerges in approximately 5% of infected cattle after a prolonged asymptomatic phase. However, current diagnostic tools, such as serological assays and PCR detection of proviral DNA from whole blood, often lack the sensitivity and specificity needed to distinguish EBL from non-EBL cases, especially in early clinical stages. This diagnostic gap emphasizes the need for reliable, non-invasive biomarkers that reflect disease progression with greater precision. Given the increased turnover of malignant cells in EBL, we investigated the potential of plasma-derived circulating cell-free DNA (cfDNA) as a novel biomarker. Proviral loads (PVL) in whole blood and plasma were quantified using qPCR targeting the BLV LTR and pol regions in cattle at different clinical stages. Although PVL in whole blood was generally higher in EBL cases, significant overlap with non-EBL animals limited its diagnostic reliability. In contrast, BLV-derived cfDNA in plasma was significantly elevated in clinically diagnosed EBL cattle and effectively distinguished between disease states. These findings highlight cfDNA as a promising tool for liquid biopsy-based approaches in veterinary virology, with the potential to enhance early diagnosis, surveillance, and control of BLV-associated malignancies in livestock populations.