Abtract: Objective　To investigate the expression levels of glypican 3 (GPC3) and its association with liver fibrosis in children with biliary atresia (BA). Methods　A total of 41 infants with BA (BA group), 9 infants with choledochal cyst (CC group) and 12 healthy infants with normal liver function and no other hepatobiliary diseases by physical examination (NC group) were collected. Serum GPC3 levels were measured by enzyme-linked immunosorbent assay (ELISA) in the 3 groups. The receiver operating characteristic (ROC) curve was used to evaluate the auxiliary diagnostic value of GPC3 for BA. Liver tissue samples from the BA group and the CC group were retained to analyze the relationship between GPC3 and the grade of liver fibrosis in children with BA by measuring the expression levels of GPC3 by immunohistochemical staining and qPCR. Results　(1) GPC3 levels were significantly higher in the BA group than those in the CC group and the NC group (χ2=24.170, P＜0.01). There were no significant differences in the serum levels of GPC3 in children aged≤30 d (n = 21) and children aged＞30 d (n = 20) in the BA group, but which were higher than those in the CC group and the NC group (χ2=24.210, P＜0.01). There were no significant differences in GPC3 levels between grades Ⅰ to Ⅱ and grades Ⅲ to Ⅳ children in the BA group, but which were higher than those in the CC group and the NC group (χ2=24.390, P＜0.01). The area under the ROC curve (AUC) of GPC3 for the diagnosis of BA was 0.878 (95%CI: 0.792-0.965, P＜0.01), with a sensitivity of 82.93%, a specificity of 80.95%, and an optimal cut-off value of 0.639 μg/L. (2) The results of liver tissue immunohistochemistry showed that GPC3 expression in liver tissue was significantly increased in the BA group compared to that of the CC group (Z=3.565, P＜0.01), and the content of GPC3 in liver tissue was positively correlated with the grade of liver fibrosis (rs=0.619, P＜0.01). The GPC3 expression was upregulated in the BA group compared to that of the CC group (Z=7.361, P＜0.05). Conclusion　The serum GPC3 has clinical value in the diagnosis and differential diagnosis of BA. GPC3 expression is upregulated in BA liver tissue, and its expression level is positively correlated with the grade of liver fibrosis in biliary atresia.