The　determination　of　folate　receptor　(FR)　that　over　expressed　in　vast　quantity　of　cancerous　cells　frequently　is　significant　for　the　clinical　diagnosis　and　treatment　of　cancers.　Many　DNA-based　electrochemical　biosensors　have　been　developed　for　FR　detection　with　high　selectivity　and　sensitivity,　but　most　of　them　need　complicated　immobilization　of　DNA　on　the　electrode　surface　firstly,　which　is　tedious　and　therefore　results　in　the　poor　reproducibility.　In　this　study,　a　simple,　sensitive,　and　selective　electrochemical　FR　biosensor　in　cancer　cells　has　been　proposed,　which　combines　the　advantages　of　the　convenient　immobilization-free　homogeneous　indium　tin　oxide　(ITO)-based　electrochemical　detection　strategy　and　the　high　selectivity　of　the　terminal　protection　of　small　molecule　linked　DNA.　The　small　molecule　of　folic　acid　(FA)　and　an　electroactive　molecule　of　ferrocence　(Fc)　were　tethered　to　3＇-　and　5＇-end　of　an　arbitrary　single-stranded　DNA　(ssDNA),　respectively,　forming　the　FA-ssDNA-Fc　complex.　In　the　absence　of　the　target　FR,　the　FA-ssDNA-Fc　was　degraded　by　exonuclease　I　(Exo　I)　from　3＇-end　and　produced　a　free　Fc,　diffusing　freely　to　the　ITO　electrode　surface　and　resulting　in　strong　electrochemical　signal.　When　the　target　FR　was　present,　the　FA-ssDNA-Fc　was　bound　to　FR　through　specific　interaction　with　FA　anchored　at　the　3＇-end,　effectively　protecting　the　ssDNA　strand　from　hydrolysis　by　Exo　I.　The　FR-FA-ssDNA-Fc　could　not　diffuse　easily　to　the　negatively　charged　ITO　electrode　surface　due　to　the　electrostatic　repulsion　between　the　DNA　strand　and　the　negatively　charged　ITO　electrode,　so　electrochemical　signal　reduced.　The　decreased　electrochemical　signal　has　a　linear　relationship　with　the　logarithm　of　FR　concentration　in　range　of　10　fM　to　10　nM　with　a　detection　limit　of　3.8　fM　(S/N=3).　The　proposed　biosensor　has　been　applied　to　detect　FR　in　HeLa　cancer　cells,　and　the　decreased　electrochemical　signal　has　a　linear　relationship　with　the　logarithm　of　cell　concentration　ranging　from　100-10000　cell/mL.　Compared　with　the　traditional　heterogeneous　electrochemical　FR　biosensors,　the　proposed　biosensor　owns　the　merits　of　the　simplicity　and　high　specificity,　presenting　the　great　potential　application　in　the　area　of　early　diagnosis　of　cancers.　(C)　2016　Elsevier　B.V.　All　rights　reserved.