Synchrotron X-ray imaging technique has been used to investigate biofluid flows in a non-destructive manner. This study aims to investigate the feasibility of the X-ray PIV technique with CO2 microbubbles as flow tracer for measurement of pulsatile blood flows under in vivo conditions. The traceability of CO2 microbubbles in a pulsatile flow was demonstrated through in vitro experiment. A rat extracorporeal bypass loop was used by connecting a tube between the abdominal aorta and jugular vein of a rat to obtain hemodynamic information of actual pulsatile blood flows without changing the hemorheological properties. The decrease in image contrast of the surrounding tissue was also investigated for in vivo applications of the proposed technique. This technique could be used to accurately measure whole velocity field information of real pulsatile blood flows and has strong potential for hemodynamic diagnosis of cardiovascular diseases.