A simple and rapid sensing strategy was proposed for chloramphenicol (CAP) detection based on structure-switching signaling aptamers. In this protocol, the aptamer can bind to both the fluorophore (FAM)-labeled complementary strand and the quencher (BHQ1)-labeled complementary strand, thus leading to the effective quenching of FAM fluorescence by BHQ1. However, when CAP is present, the structure switch is reversed because the aptamer recognizes CAP, resulting in fluorescence recovery. Such a fluorescence-sensing platform can monitor CAP within a good linear range (1-100 ng/mL), with a detection limit of 0.70 ng/mL. Cross-reactivity with other common antibiotics is negligible, indicating the excellent selectivity of the strategy. Moreover, as the aptamers are not modified, this method is simple and low-cost. The present work reveals a new direction for detecting CAP or other target compounds without prior knowledge of the secondary or tertiary structures of the aptamer.