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Abstract Staphylococcus aureusis a major foodborne bacterial pathogen. Early detection ofS. aureusis crucial to prevent infections and ensure food quality. The iron‐regulated surface determinant protein A (IsdA) ofS. aureusis a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in‐silico approach to develop and validate high‐affinity binding aptamers for the IsdA protein detection using custom‐designed in‐silico tools and single‐molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in‐silico oligonucleotide screening methods and metadynamics‐based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA‐aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in‐silico approach to support aptamer discovery. This study showcases a computational SELEX method in combination with single‐molecule binding studies deciphering effective aptamers againstS. aureus IsdA, protein. The established approach demonstrates the ability to expedite aptamer discovery that has the potential to cut costs and predict binding efficacy. The application can be extended to designing aptamers for various protein targets, enhancing molecular recognition, and facilitating the development of high‐affinity aptamers for multiple uses.