Introduction: Tissue-engineered oral mucosa (TEOM) is increasingly being used to model oral mucosal diseases and to assess drug toxicity. Current TEOM models are constructed using normal oral fibroblasts (NOF) contained within a hydrogel matrix with normal oral keratinocytes (NOK) cultured on top. NOK are not commercially available and suffer from donor-to-donor variability. Therefore, oral mucosal models based on immortalised keratinocytes may offer advantages over NOK-based models. The objective of this study was to construct and characterise the TEOM developed using TERT2-immortalised oral keratinocyte (FNB6) cells and validate its similarity to normal oral muco-sal tissue. Methods: TEOM were constructed by culturing FNB6 cells on top of a NOF-populated collagen type-1 hydrogel in tissue culture transwell inserts cultured at an air-to-liquid interface and collected at 14 day. TEOM were subjected to morphological (H&E and PAS), ultrastructural (TEM) and immunohistological (Ki-67, cytokeratin 14 and E-cadherin) analysis. Results: Histologically TEOM mimicked native oral mucosa displaying a stratified epithelium, fibroblast-containing connective tissue and basement membrane. Furthermore, TEM confirmed the presence of des-mosomes and hemi-desmosomes in the epithelium. IHC revealed expression of differentiation markers (cytokeratin 14), proliferation (Ki-67), cell adhesion (E-cadherin). Conclusion: FNB6 mucosal models able to mimic native oral mucosa structure. It has potential for drug delivery and toxicity evaluation, and replacing models based on NOK where access to primary cells is limited.