This study aimed to evaluate the feasibility and repeatability of the flash-replenishment method in contrast-enhanced ultrasound (CEUS) perfusion imaging and assess quantitatively microvascular perfusion in the liver. Twenty healthy New Zealand rabbits were submitted to CEUS perfusion imaging with continuous intravenous infusion. Using flash-replenishment kinetics, the dynamic process of depletion and refilling of microbubble contrast agent was recorded. The hepatic microvascular perfusion parameters were calculated, including region of interest, peak intensity (PI), area under the curve (AUC), and hepatic artery to vein transit time (HA-HVTT). A consistency test was performed for multiple measurements by the same operator and blind measurements by two different operators. The hepatic perfusion imaging of 3×108 bubbles/min had minimal error and the best imaging effect and repeatability. The variability of the perfusion parameter measured at 3 cm depth under the liver capsule was at a minimum with coefficient of variation of 3.9%. The interclass correlation coefficient (ICC) of measurements taken by the same operator was 0.985, (95% confidence interval, CI=0.927-0.998). Measurements taken by two operators had good consistency and reliability, with the ICC of 0.948 (95%CI=0.853-0.982). The PI and AUC of liver parenchyma after reperfusion were lower than before blocking; and HA-HVTT was significantly longer than before blocking (P<0.05). The flash-replenishment method in CEUS perfusion imaging showed good stability and repeatability, which provide a valuable experimental basis for the quantitative assessment of hepatic microvascular perfusion in clinical practice.