Staphylococcus aureus is ranked among the top five most common foodborne pathogens affecting public health and the economy worldwide. To improve detection and reduce diagnostic burdens, several detection methods from traditional culture-based techniques to biosensing platforms have evolved. Among several markers, surface proteins are considered to be the most important markers due to the specific roles they play in the survival and colonization of the bacterium on hosts. Here, we have developed a detection platform for a key surface protein, iron-regulated surface determinant protein A (IsdA), using a combination of computationally developed aptamer and single-molecule fluorescence resonance energy transfer (smFRET). Computationally generated RNA aptamer incorporated into the FRET-based sensor show high specificity detection of IsdA with a detection limit down to 0.6 pM and dynamic range extending to ∼10 nM. Molecular dynamics (MD) simulations show distinct conformational flexibility of the unbound aptamer and a reduced flexibility for the aptamer-IsdA complex, corresponding to the experimentally observed higher FRET efficiencies. The FRET-based single-molecule aptasensor that we developed has great potential for rapid monitoring S. aureus. Further, the developed approach has the potential to be broadly applicable across diverse fields of biotechnology including environmental monitoring, forensic analysis, and clinical diagnostics.