Fish-derived collagen is considered a promising material for oral mucosa tissue engineering because it is free from risk of transmissible infectious diseases. We developed a novel fish scale collagen scaffold with micropatterns on which oral keratinocytes are cultured to create a tissue-engineered oral mucosa. Anisotropic and isotropic etching were used to fabricate negative patterns on the Si substrate and soft lithography was used to translate the patterns to PDMS and fish scale collagen. The histological evaluation of the tissue-engineered oral mucosa confirmed the successful formation of the epithelial layer mimicking oral mucosa in vivo. This study suggests that our novel approach for fabricating biomimetic scaffold is promising and will make a huge contribution to the field of oral mucosa regeneration.