A　simple,　sensitive,　and　selective　immobilization-free　electrochemical　aptasensor　had　been　developed　which　combines　the　advantages　of　the　discrimination　of　the　aggregation　of　long　and　short　DNA　on　a　negatively　charged　indium　tin　oxide　(ITO)　electrode,　high　selectivity　of　the　aptamer,　and　high　efficiency　of　exonuclease-catalyzed　target　recycling　amplification.　Ochratoxin　A　(OTA),　a　type　of　mycotoxin,　has　been　chosen　as　the　model　target.　Methylene　blue　(MB)　labeled　probe　DNA　had　been　hybridized　with　the　OTA　aptamer　first,　which　cannot　diffuse　freely　to　the　negative　charged　ITO　electrode　surface　due　to　the　repulsion　of　the　negative　charges,　since　the　hybridized　DNA　contains　large　negative　charges.　In　the　presence　of　target　(OTA),　the　aptamer　prefers　to　form　an　OTA-aptamer　complex　in　lieu　of　an　aptamer-DNA　duplex,　which　results　in　the　dissociation　of　probe　DNA　from　the　probe　DNA-aptamer　complex.　The　released　probe　DNA　could　be　digested　into　mononucleotides,　including　a　MB-labeled　electroactive　mononucleotide　(eT),　due　to　the　employment　of　the　RecJf　exonuclease,　a　single-stranded　DNA　specific　exonuclease.　Since　the　eT　contains　little　negative　charge,　it　can　diffuse　easily　to　the　negative　charged　ITO　electrode　surface,　which　results　in　the　enhanced　electrochemical　response　detected.　At　the　same　time,　the　aptamer　in　the　OTA-aptamer　complex　can　be　digested　by　RecJf　exonuclease　also　to　liberate　the　target,　which　can　participate　in　the　next　reaction　cycling　and　realize　the　electrochemical　signal　amplification.　Based　on　this　strategy,　an　ultrasensitive　homogeneous　immobilization-free　electrochemical　aptasensor　for　OTA　can　be　developed　with　a　low　detection　limit　(LOD)　of　0.004　ng　mL-1　(S/N　=　3).　The　proposed　biosensor　combines　the　advantages　of　the　simplicity　of　immobilization-free　homogeneous　ITO　based　electrochemical　determination,　high　efficiency　of　exonuclease-catalyzed　target　recycling,　and　high　selectivity　of　the　aptamer.　The　fabricated　biosensor　has　been　applied　to　detect　OTA　in　real　samples　with　satisfactory　results.　The　same　strategy　can　be　applied　to　develop　biosensors　for　diverse　targets.　©　2015　American　Chemical　Society.