OBJECTIVE To investigate perfusion change in contrast-enhanced ultrasonography (CEUS) to evaluate liver fibrosis based on biliary obstruction using an animal model. METHODS New Zealand white rabbits (3-4 kg) underwent bile duct ligation to form a biliary obstruction model. We performed liver CEUS and laboratory tests on the day before the operation (day 0) and every 7 postoperative days until the rabbits were sacrificed. After CEUS, signal intensity of liver parenchyma with a time-intensity curve was analyzed. Perfusion parameters were automatically calculated from region-of-interests, including peak signal intensity, mean transit time, area under the curve and time to peak. Histological grades of liver fibrosis were assessed according to the Metavir score system immediately after sacrifice. Generalized estimating equations were used to analyze the association between liver fibrosis grades and perfusion parameters for statistical analysis. The perfusion parameters were measured on the last day and the difference between day 0 and the last day were evaluated. RESULTS From the nine rabbits, histological grades of liver fibrosis were grade 1 in one rabbit, grade 2 and 3 in three rabbits each, and grade 4 in two rabbits. Among the four CEUS parameters, only the peak signal intensity measured on the last day demonstrated a significant association with liver fibrosis grades (OR = 1.392, 95%CI: 1.114-1.741, P = 0.004). The difference in peak signal intensity between day 0 and the last day also demonstrated an association with liver fibrosis (OR = 1.191, 95%CI: 0.999-1.419, P = 0.051). The other parameters tested, including mean transit time, area under the curve, and time to peak, showed no significant correlation with liver fibrosis grades. CONCLUSIONS This animal study demonstrates that CEUS can be used to evaluate liver fibrosis from biliary obstruction using peak signal intensity as a parameter.