Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease. This study sought to engineer a bilayered collagen-hyaluronate (CHyA-B) scaffold for the development of a physiologically-representative 3D in vitro tracheobronchial epithelial co-culture model. CHyA-B scaffolds were fabricated by integrating a thin film top-layer into a porous sub-layer with lyophilisation. The film layer firmly connected to the sub-layer with delamination occurring at stresses of 12e15 kPa. Crosslinked scaffolds had a compressive modulus of 1.9 kPa and mean pore diameters of 70 mm and 80 mm, depending on the freezing temperature. Histological analysis showed that the Calu-3 bronchial epithelial cell line attached and grew on CHyA-B with adoption of an epithelial monolayer on the film layer. Immunofluorescence and qRT-PCR studies demonstrated that the CHyA-B scaffolds facilitated Calu-3 cell differentiation, with enhanced mucin expression, increased ciliation and the formation of intercellular tight junctions. Co-culture of Calu-3 cells with Wi38 lung fibroblasts was achieved on the scaffold to create a submucosal tissue analogue of the upper respiratory tract, validating CHyA-B as a platform to support co-culture and cellular organisation reminiscent of in vivo tissue architecture. In summary, this study has demonstrated that CHyA-B is a promising tool for the development of novel 3D tracheobronchial co-culture in vitro models with the potential to unravel new pathways in drug discovery and drug delivery.