Biliary atresia (BA) is a poorly understood deadly liver disease, often diagnosed late, is incurable and frequently requires liver transplantation. In this study, we aim to investigate the underlying pathomechanisms and molecular signatures associated with BA.We combine organoid and transcriptomic analysis to gain new insights into BA pathobiology.Liver organoids from BA patients and a BA mouse model, Rhesus rotavirus A-infected mice, exhibited aberrant morphology and disturbed apical-basal organization. Transcriptomic analysis of BA organoids revealed a shift from cholangiocyte to hepatocyte transcriptional signatures and altered beta-amyloid-related gene expression. Beta-amyloid accumulation was observed around the bile ducts in BA livers and exposure to beta-amyloid induced the aberrant morphology in control organoids.Beta-amyloid deposition represents a novel finding with pathobiological implications, a new diagnostic feature and uncovers a previously unsuspected disease mechanism for BA.Biliary atresia (BA) is a poorly understood and devastating obstructive bile duct disease of newborns, often diagnosed late, is incurable and frequently requires liver transplantation. Using human and mouse 'liver mini-organs in the dish', we unexpectedly identified beta-amyloid deposition, the main pathological feature of Alzheimer's disease and Cerebral Amyloid Angiopathy, around BA bile ducts. This finding reveals a novel pathomechanism for BA important for future diagnosis and treatment considerations.