Biomimetic scaffolds mimic important features of the extracellular matrix (ECM) architecture and can be finely controlled at the nano- or microscale for tissue engineering. Rational design of biomimetic scaffolds is based on consideration of the ECM as a natural scaffold; the ECM provides cells with a variety of physical, chemical, and biological cues that affect cell growth and function. There are a number of approaches available to create 3D biomimetic scaffolds with control over their physical and mechanical properties, cell adhesion, and the temporal and spatial release of growth factors. Here, an overview of some biological features of the natural ECM is presented and a variety of original engineering methods that are currently used to produce synthetic polymer-based scaffolds in pre-fabricated form before implantation, to modify their surfaces with biochemical ligands, to incorporate growth factors, and to control their nano- and microscale geometry to create biomimetic scaffolds are discussed. Finally, in contrast to pre-fabricated scaffolds composed of synthetic polymers, injectable biomimetic scaffolds based on either genetically engineered- or chemically synthesized-peptides of which sequences are derived from the natural ECM are discussed. The presence of defined peptide sequences can trigger in situ hydrogelation via molecular self-assembly and chemical crosslinking. A basic understanding of the entire spectrum of biomimetic scaffolds provides insight into how they can potentially be used in diverse tissue engineering, regenerative medicine, and drug delivery applications.