The determination of folate receptor(FR)that over expressed in most of cancerous cells frequently is significant for the clinical diagnosis and treatment of cancers.[1] Herein,a simple but sensitive and selective electrochemical FR biosensor in cancer cells has been proposed,which combines the advantages of the convenient immobilization-free homogeneous 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 respectively tethered to 3end and at 5end of a single-stranded DNA(ssDNA),forming the FA-ssDNA-Fc complex.In the absence of FR,the FA-ssDNA-Fc can be degraded by exonuclease I(Exo I)from 3end and produced a free Fc,which can diffuse freely to the ITO electrode surface and results in strongly electrochemical signal.However,when FR was present,the FA-ssDNA-Fc can be bound to FR through specific interaction with FA anchored at the 3-end,which effectively protected the ssDNA from hydrolysis by Exo I.Thus the FR-FA-ssDNA-Fc cannot diffuse easily to ITO electrode surface due to the electrostatic repulsion between the DNA strand and the negative charged ITO electrode and electrochemical signal was reduced.Then the electrochemical FR biosensor can be established and has been applied to detect FR in HeLa cancer cells based on this principle.