It　is　important　to　develop　clinical　point-of-care　testing　(POCT)　methods　to　avoid　side　effects　and　even　fatalities　due　to　anesthetic　drug　overdose.　Due　to　their　inherent　advantages　(e.g.　ease　of　modification,　thermal　stability,　and　low　cost),　electrochemical　aptamer-based　(EAB)　sensors　are　promising　alternatives　to　conventional　analytical　methods.　However,　most　of　the　current　methods　require　some　complex　modification　steps　for　the　preparation　of　electrodes　or　signal　probes　as　well　as　the　indirect　measurement　of　signals,　and　the　application　of　these　methods　as　POCT　devices　remains　a　challenge.　In　this　work,　a　sandwich-type　EAB　sensor　was　developed　for　the　direct　determination　of　procaine　in　biofluids.　The　original　aptamer　was　split　into　two　segments,　with　one　fragment　modified　with　thiol　and　the　other　modified　with　a　redox　label.　The　detection　process　was　rapid　as　conformational　changes　occur　almost　instantaneously　when　the　aptamer　binds　to　the　target.　Moreover,　the　binding　properties　of　the　aptamer　towards　the　target　were　characterized　by　circular　dichroism　spectroscopy,　molecular　docking　simulations　and　apparent　electron　transfer　rates　test.　The　simple,　cost-effective　and　reagentless　sandwich-type　EAB　sensor　achieved　nanomolar　detection　of　procaine　with　a　limit　of　detection　(LOD)　of　1.9　nM,　5.4　nM　and　3.3　nM　in　buffer,　5　%　urine　and　2.5　%　serum,　respectively.　This　work　is　expected　to　play　a　role　in　the　field　of　clinical　drug　analysis.　©　2025　Elsevier　B.V.