Supramolecular hydrogels constructed through molecular self-assembly of small molecules have unique stimuli-responsive properties; however, they are mechanically weak in general, relative to conventional polymer gels. Very recently, we developed a zwitterionic amino acid tethered amphiphilic molecule , which gave rise to a remarkably stiff hydrogel comparable with polymer-based agarose gel, retaining reversible thermal-responsive properties. In this study, we describe that rational accumulation of multiple and orthogonal noncovalent interactions in the supramolecular nanofibers of played crucial roles not only in the mechanical reinforcement but also in the multistimuli responsiveness. That is, the zwitterionic amino acid moiety and the CC double bond unit of the hydrogelator can function as a pH-responsive unit and a light-responsive unit, respectively. We also demonstrated that this stiff and multistimuli-responsive supramolecular hydrogel is applied as a unique mold for 2D and 3D-patterning of various substances. More significantly, we succeeded in the fabrication of a collagen gel for spatial patterning, culturing, and differentiation of live cells by using hydrogel molds equipped with 2D/3D microspace channels (100–200 μm in diameter).