A novel strategy for the quantitative determination of human colon cancer DLD-1 cells utilizing an electrochemical aptasensor was developed by effective surface recognition between Mucin 1 glycoprotein over-expressed on the cell membrane and MUC-1 aptamer (MUC-1) bound on carbon nanospheres (CNSs). An MTT assay revealed that the as-prepared CNSs by green route exhibited satisfactory biocompatibility for cell viability, providing a suitable platform for the cell adhesion study. Furthermore, using CNSs as a sensing layer accelerated electron transfer and provided a highly stable matrix for the convenient conjugation of target MUC-1 aptamer, considerably amplifying the electrochemical signals. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were applied to assess the optimal conditions and detection performance of the as-fabricated aptasensor. The attachment of colon cancer DLD-1 cells onto the MUC-1 aptamer immobilized CNSs led to increased EIS responses, which changed linearly in cell concentration ranging from 1.25 × 10(2) to 1.25 × 10(6) cells per mL with a lower detection limit of 40 cells per mL. With this method, colon cancer DLD-1 cells can be easily distinguished from normal cells, Human astrocytes 1800. The novel aptasensor revealed high specificity to DLD-1 cells. Furthermore, the aptasensor described here showed good reproducibility and high stability because of the CNSs of high stability and biocompatibility. The proposed protocols are a promising technique for the early monitoring of human colon cancer, and might have potential clinical applications such as cancer diagnosis, drug screening.