Exosomes are considered potential biomarkers for early screening and accurate non-invasive diagnosis of cancer, so development of innovatively facile approaches for the detection of cancer cell-derived exosomes has become increasingly important. Herein, we propose a facile electrochemical biosensor based on divalent aptamer-functionalized nanochannels for highly efficient detection of cancer cell-derived exosomes. The aptamer against transmembrane receptor protein CD63 and the aptamer targeting membrane protein EpCAM are simultaneously immobilized on the nanochannels to construct the divalent aptamer-functionalized nanochannels. Thus, the target exosomes can be recognized and selectively captured by the functionalized nanochannels in a divalent collaborative manner. The combined exosomes overlay the ion channel effectively and hinder the ionic flow through the nanochannels, resulting in an evidently varied ionic transport behavior corresponding to the abundance of exosomes. The divalent aptamer-functionalized nanochannels can substantially promote the binding stability and enhance the detection specificity, while the sensitivity of detection is improved greatly by virtue of the amplified response of array channels synergized with the electrochemical technique. Therefore, the developed biosensor provides a highly specific, sensitive, and accurate approach for the detection of cancer cell-derived exosomes, which may hold great potential for application in early clinical cancer diagnosis.