Single‐molecule optical and electrical measurements of transmembrane ion channels can reveal disparate behavioral subpopulations that are not detectable via conventional approaches. However, such measurements require fluorescently labeled materials of high purity and functional integrity. We produced eight mutants of Staphylococcus aureus α‐hemolysin (αHL), introducing single cysteine residues at strategic locations on the extracellular cap of the protein. Expression in E. coli and purification by affinity chromatography were optimized to produce protein with greater than 95% purity at concentrations of more than 1 mg/ml. Mutant proteins were derivatized with one of two fluorescent labels (BODIPY‐TMR or TMRIA) followed by proteolytic removal of the affinity tag and further purification. The resulting fluorescently labeled αHL proteins are devoid of contaminating free dye and demonstrate pore‐forming capacity in planar lipid membranes.