To explore the potential of step-by-step assembly in the fabrication of biological materials, we designed and synthesized two peptide-based molecules for enzyme-instructed hierarchical assembly. Upon the treatment of alkaline phosphatase, one molecule undergoes enzyme-instructed self-assembly forming uniformed nanofibers. The other one that can self-assemble into vesicles undergoes enzyme-induced transformation of self-assembly converting vesicles into irregular aggregates upon the treatment of carboxylesterase. Coadministration of two enzymes to a mixture of these two molecules in a stage-by-stage fashion leads to a physically knotted nanofibrous scaffold that is applicable as a nanostructured matrix for cell culture.