Salivary alpha-amylase (sAA) is a stress biomarker in human diseases, but there are no reports of sAA measurements in diseased dogs. This study measured the sAA and serum alpha-amylase (AA) levels in 16 healthy dogs and 31 diseased dogs using a kinetic enzyme assay to assess the stress status. The sAA and serum AA levels were significantly higher in the diseased dogs than in healthy dogs (p < 0.05), but there was no correlation between the 2 groups (r = 0.251, p = 0.089). This suggests that sAA can be useful as a stress biomarker in diseased dogs.