Small-molecule-induced assembly of defined protein structures could have broad implications for the fabrication of new materials as well as biological signaling pathways. However, the design of new hostguest pairs capable of small-molecule-induced assembly in a biologically relevant context remains a significant challenge. Herein, we report a series of miniprotein hosts, evolved from the tenth type III domain of fibronectin (Fn3), that display remarkable binding affinity toward a red-shifted environment-sensitive merocyanine derivative, termed sI-Pht. Importantly, the consensus binder isolated from directed evolution experiments (6.2.18) forms a higher order assembly in response to addition of sI-Pht, as assessed by analytical ultracentrifugation. sI-Pht-induced assembly of 6.2.18 results in a 570-fold increase in fluorescence compared to free dye. This property enables the direct visualization of hostguest assemblies by fluorescence microscopy. As a demonstration, we show that supramolecular assembly of the 6.2.18-sI-Pht system can be visualized on the surface of living yeast cells. This new hostguest pair provides a tool for the potential development of new materials as well as pathway engineering. In a broader context, this work details a new design paradigm for the discovery of hostguest systems that function in the context of living cells.