Objectives: The present study aimed to estimate the value of serum interleukin-33 (IL-33) levels in infants with cholestasis, correlate serum IL-33 levels with the clinicopathological profile of infants with cholestasis, and compare its level with that of healthy infants who served as control. Methods: Sixty infants with cholestasis were enrolled in the present study and divided into biliary atresia (BA) group and non-BA group, in addition to 30 healthy infants as a control group. All infants were analyzed for their clinical and biochemical features, histopathological profile, and serum level of IL-33 by enzyme-linked immune sorbent assay. Results: Serum level of IL-33 in BA group (median 48.0, interquartile range: 28.9–106.2) was significantly higher than that of the non-BA group (median 17.3, interquartile range: 13.7–18.8 pg/mL) and both were higher than that of the control group. There was a positive correlation between serum IL-33 and aspartate aminotransferase, alanine aminotransferase, bilirubin (total and direct) levels, and fibrosis stage among the BA group. Serum IL-33 at a cut-off value of 20.8 pg/mL can detect BA with a specificity of 95% and a sensitivity of 96.7%. Conclusion: The significantly higher production of IL-33 in patients with BA compared to non-BA suggests a potential role of IL-33 for initiation and progression of the disease process, also, IL-33 may have a diagnostic role in infants with BA.