The widespread use of sulfonamide (SA) antibiotics in animal husbandry has led to residues of SAs in the environment, causing adverse effects to the ecosystem and a risk of bacterial resistance, which is a potential threat to public health. Therefore, it is highly desirable to develop simple, high-throughput methods that can detect multiple SAs simultaneously. In this study, we isolated aptamers with different specificities based on a multi-SA systematic evolution of ligands by the exponential enrichment (SELEX) strategy using a mixture of sulfadimethoxine (SDM), sulfaquinoxaline (SQX), and sulfamethoxazole (SMZ). Three aptamers were obtained, and one of them showed a similar binding to all tested SAs, with dissociation constant () ranging from 0.22 to 0.63 μM. For the other two aptamers, one is specific for SQX, and the other is specific for SDM and sulfaclozine. A label-free detection method based on the broad-specificity aptamer was developed for the simultaneous detection of six SAs, with detection of limits ranging from 0.14 to 0.71 μM in a lake water sample. The aptasensor has no binding for other broad-spectrum antibiotics such as β-lactam antibiotics, quinolones, tetracyclines, and chloramphenicol. This work provides a promising biosensor for rapid, multiresidue, and high-throughput detection of SAs, as well as a shortcut for the preparation of different specific recognition elements required for the detection of broad-spectrum antibiotics.