Serum thymidine kinase (TK) activity has recently been evaluated as a serum marker for human and canine hematopoietic neoplasms. The purpose of the current study was to establish the significance of serum TK activity in the diagnosis of bovine leukosis. The discrimination value for TK activity was set at 5.4 U/l based on the receiver operating characteristic curve. In the group of clinically healthy cows, only 2 out of 83 cows (2.4%) had serum TK activity above the discrimination value. In contrast, 19 out of 20 cows (95.0%) with bovine leukosis showed serum TK activity above the discrimination value, although only 7 of 79 (8.9%) cows diagnosed with diseases other than bovine leukosis showed elevated serum TK activity. Thymidine kinase activities of all Bovine leukemia virus-positive cows with or without lymphocytosis were below the discrimination value. Sensitivity and specificity of measuring serum TK activity as a diagnostic tool for bovine leukosis was 95.0% and 95.9%, respectively. Results indicate that serum TK activity may be a marker for bovine leukosis.