Sulfonamides　(SAs)　is　a　class　of　antibiotics　that　extensively　used　for　treating　infectious　diseases　in　livestock　industries　and　aquaculture.　Thus,　it　is　urgent　need　to　obtain　the　bio-receptor,　which　has　excellent　cross-reactivity　and　specificity　to　SAs,　for　developing　high-throughput　methods　for　the　determination　of　multiple　SAs　even　all　commonly-used　SAs,　to　realize　the　quick　screening/detection　of　total　SAs　in　animal-derived　foods.　We　herein　isolated　several　SAs-specific　cross-reactive　aptamers　by　using　a　library-immobilized　SELEX　with　multi-SAs　parallel　selection　strategy.　Two　of　the　isolated　aptamers　(Sul-01　and　Sul-04)　can　specifically　recognize　and　bind　seven　SAs　respectively　with　higher　binding　affinity　and　no　interference　of　non-sulfonamide　antibiotics,　and　thus　can　be　applied　as　bio-receptors　for　developing　high-throughput　aptasensors　for　the　quick　screening/detection　of　multiple　SAs.　By　using　the　mixture　of　Sul-01　and　Sul-04　as　bio-receptor,　a　ratiometric　fluorescent　aptasensor　was　created　for　the　quick　detection　of　nine　SAs　including　sulfamethoxydiazine　(SMD),　sulfapyridine　(SPD),　sulfaquinoxaline　(SQ),　sulfathiazole　(ST),　sulfamonomethoxine　(SMM),　sulfamerazine　(SMR),　sulfaguanidine　(SG),　sulfamethazine　(SMZ)　and　sulfadiazine　(SD)　with　a　detection　limit　(LOD)　of　0.10–0.50　μM,　or　total　of　above　nine　SAs　with　a　LOD　of　0.20　μM.　The　fluorescent　aptasensor　was　successfully　applied　to　detect　each　or　total　of　SMD,　SPD,　SQ,　ST,　SMM,　SMR,　SG,　SMZ　and　SD　in　fish　samples　with　a　recovery　of　83　%–92　%　and　a　relative　standard　deviation　(RSD,　n　=　5)　©　2024　Elsevier　B.V.