Contrast-enhanced ultrasonography has an important role in the detection of tumors in humans. The second-generation contrast agent Sonazoid has the ability of real-time contrast imaging along with parenchymal imaging. The purposes of this study were to determine the effect and duration of Sonazoid on the changes in gray-scale enhancement of canine spleen and to establish an appropriate protocol for contrast-enhanced ultrasonography of canine spleen. Six healthy beagles were injected with an intravenous bolus of Sonazoid. In the spleen parenchyma, the enhancement was maintained up to 30 min after injection. Moreover, for 5–22 s after injection, gray-scale enhancement of splenic arteries afforded arterial imaging. Perfusion of the kidney may be investigated from 3.6 s to 3.5 min after injection of Sonazoid. These results suggest that Sonazoid is applicable to canine spleen parenchymal imaging and that the optimal time for the parenchymal imaging is 7–30 min after injection. The findings of this quantitative study should prove useful in the evaluation of diffuse or focal splenic and renal diseases in dogs.