Current artificial tissue‐substitutes have limited clinical applications due to unmatched complex combination of cells and extracellular matrix (ECM) as seen in native tissues. From a developmental perspective, the construction of effective biomimetic tissues is from the bottom (one‐dimensional nanoparticles or two‐dimensional membranes) up (three‐dimensional scaffolds or more complex composite). In a hierarchical architecture, each sub‐structure can be assembled in a flexible way with specific regulators and cells, which overcomes the deficiency of one‐for‐all scaffold. Here, a cell‐compatible cell‐lined layered nano‐membrane is developed. Bioactive molecules are mounted on a nano‐membrane and later released to its lined cell sheet. The cell‐lined membrane is in a free‐standing form to regulate cellular functions. The major advantage of this methodology is to provide a versatile approach to construct biomimetic tissues for clinical applications.