Verotoxin-producing Escherichia coli (VTEC) present a significant risk of foodborne illness and severe patient outcomes. Isolation is laborious and time consuming due to the diversity of strains. Development of a differential agent would enhance rate and speed of isolation. Production of verotoxin (VT) is the only phenotypic trait exclusive to VTEC. Aptamers are single-stranded oligonucleotides with high selectivity and affinity for their targets. A VT-targeting aptamer beacon could aid in isolation of VTEC. Proof of concept was demonstrated using an existing aptamer sequence, showing increased fluorescence in the presence of VT1a. Further characterization showed target specificity, but weaker signal contrast in complex media. Comparison of fluorophore-quencher pairs showed Texas Red and Black Hole Quenchers as optimal choices. Finally, a DNA substrate developed to detect VT enzyme activity produced weak fluorescent signal making it unlikely to aid in detection.