The care of patients with cholestatic liver diseases such as primary sclerosing cholangitis (PSC) is challenging, partly due to the lack of knowledge of disease pathogenesis and suitable in vitro models for disease modeling and drug screening. Although the pathogenesis of cholestatic liver diseases like PSC remains unknown, the importance of the vascular-biliary interface is clear. Cholangiocyte injury not only impairs barrier function such that bile leaks and damages periductal tissue, but also activates cholangiocytes to secret pro-inflammatory and pro-fibrogenic mediators to stimulate immune cells and mesenchymal cells, ultimately causing damage to the liver. Here we describe a detailed protocol for fabricating a human vascularized bile duct-on-a-chip (VBDOC) that consists of a vascular channel, biliary channel, and neighboring mesenchymal cells in a collagen gel that models the vascular-biliary interface structurally and functionally in three dimensions. This device is notable in maintaining cholangiocyte polarity and barrier function, recapitulating physiological functions and responses of the large bile ducts, and enabling manipulation of components of the mechanical microenvironment such as matrix stiffness and shear flow in the lumens. This practical workflow could help researchers manufacture the VBDOC in their own labs and apply it to studies of various cholestatic liver diseases.