Spontaneous, highly ordered large-scale fibronectin networks driven by electrostatic polymer patterns are fabricated, and these precisely controlled protein connections are demonstrated. It is examined whether this scheme, universal to various fibrillar extracellular matrix proteins beyond fibronectin, collagen, and laminin, can be self-organized. These data reveal a novel bottom-up method to form anisotropic, free-standing protein networks to be used as flexible, transferrable substrates for cardiac and neuronal tissue engineering.