In recent years, staphylococcal food poisoning (SFP) caused by the ingestion of food contaminated with Staphylococcus aureus enterotoxins (SEs) has been one of the most common foodborne illnesses worldwide. As a result, rapid, sensitive and reliable detection methods are crucial for routine observations of SEs. However, current detection methods are primarily antibody-dependent, and the development of methods is limited by the preparation and instability of fresh antibodies during testing. In this study, aptamers that bind to Staphylococcus aureus enterotoxin A (SEA) with high affinity and selectivity were generated in vitro by a twelve-round selection process based on magnetic separation technology with a dissociation constant (K_d) value as low as 48.57 ± 6.52 nM. The optimal aptamer A15 was successfully used in the fluorescent bioassay to detect SEA in the food sample with a detection limit of 8.7 × 10⁻³ μg mL⁻¹. Based on this study, the selected aptamers can be expected to be new molecular recognition elements that can be used in innovative biosensors for SEA detections.