Collagen, a key structural protein in the extracellular matrix (ECM), provides essential physical and biological support for cells, making it indispensable in tissue engineering (TE). Producing collagen‐mimicking polypeptides (CMPs) in E. coli offers advantages such as rapid production, cost efficiency, and ease of genetic modifications. These CMPs can self‐assemble into collagen fibrils, although they lack the post‐translational modifications (PTMs) required for structural stability. To address this, the described E. coli system employs genetic code expansion to incorporate L‐3,4‐dihydroxyphenylalanine (L‐DOPA) into CMPs at specific sites. The catechol side chain of L‐DOPA enhances molecular structural stability, supports cellular attachment, and promotes cell growth. These CMPs form a triple‐helix structure and self‐assemble in vitro to construct collagen fibrils, with the inclusion of L‐DOPA significantly enhancing the fibrillation process. The CMPs are biocompatible, enabling the spreading and increased metabolic activity of human fibroblasts cultured on 2‐D hydrogels or within 3‐D scaffolds, contingent on the presence of L‐DOPA‐incorporated CMPs. This system allows for precise genetic modifications, incorporating non‐canonical amino acids to customize CMP properties for diverse TE applications. This innovative strategy merges TE and synthetic biology to improve collagen‐based biomaterials, providing custom‐made solutions.