The goal of this study was to evaluate the suitability of a commercially available D-dimer assay as a diagnostic tool for testing dogs. This assay is an immunoturbidimetric diagnostic test, capable of determining the D-dimer levels in human plasma by using 2B9 monoclonal antibody. Plasma samples of clinically healthy (n = 20) and tumour-bearing (n = 50) dogs were measured. The tumours were grouped on the basis of histological type and aggressiveness, and then the measured D-dimer concentrations of these groups were compared to those of the control group. The differences were analysed statistically. For benign tumours, we did not find alterations in the D-dimer levels. However, in the case of malignant tumours (lymphoma, sarcoma, and carcinoma) and in the presence of metastases, significantly elevated D-dimer levels were measured. The assay proved to be suitable for measuring the D-dimer levels in plasma samples of dogs. The calculated reference range for dogs was confirmed to be between 0.06 and 0.69 µg/mL fibrinogen equivalent unit.