In terms of the characteristic of exonuclease I that can selectively catalyze the degradation of single-stranded DNA, we report a new sensitive electrochemical biosensor for the detection of breast cancer cells by using the aptamers of MUC1 that is overexpressed on the surface of MCF-7 cells. Because the binding of aptamer and MUC1 that is over-expressed on the surface of breast cancer cell MCF-7 can prevent its hybridization with the complementary probe DNA, the unhybridized single-stranded probe DNA that is immobilized on the electrode surface will be selectively digested by Exonuclease I and result in the removal of the signal molecule methylene blue. Therefore, by tracing the changes in electrochemical responses, the biosensor can be used to sensitively detect MCF-7 cells in a linear range from 103 to 106 cell/mL with a detection limit of 330 cell/mL, and easily distinguish the control islet β cells with high specificity.