KcsA, a potassium channel from Streptomyces lividans, is one of the most extensively studied transmembrane proteins. Despite significant research in structural biology, relatively few ligands of KcsA have been identified. One such ligand is Hui1, an artificial peptide derived from a phage display screening using a combinatorial library constructed relying on several sea anemone toxins. In this study, we engineered a fluorescent probe by fusing Hui1 with enhanced green fluorescent protein (eGFP), creating the first fluorescence-based tool to visualize prokaryotic ion channels. The eGFP-Hui1 chimera was successfully produced in Escherichia coli and purified using chromatographic techniques. Our study revealed a direct interaction between KcsA, also recombinantly expressed in E. coli, and the fluorescent chimera. Furthermore, we demonstrated that both Hui1 and tetraethylammonium can effectively displace the chimera from its complex with KcsA, confirming the specificity of the binding interaction. This approach opens new avenues for pharmacological and structural investigations, including the development of novel antimicrobial agents and high-throughput ligand screening.