A rapid and convenient homogeneous aptamer sensor with high sensitivity is highly desirable for the electrochemical detection of tumor biomarkers. In this work, a homogeneous electrochemical aptamer sensor is demonstrated based on a two-dimensional (2D) nanocomposite probe and nanochannel modified electrode, which can realize sensitive detection of carcinoembryonic antigen (CEA). Using π-π stacking and electrostatic interaction, CEA aptamer (Apt) and cationic redox probe (hexaammineruthenium(III), Ru(NH3)63+) are co-loaded on graphite oxide (GO), leading to a 2D nanocomposite probe (Ru(NH3)63+/Apt@GO). Vertically ordered mesoporous silica-nanochannel film (VMSF) is easily grown on the supporting indium tin oxide (ITO) electrode (VMSF/ITO) using the electrochemical assisted self-assembly (EASA) method within 10 s. The ultrasmall nanochannels of VMSF exhibits electrostatic enrichment towards Ru(NH3)63+and size exclusion towards 2D material. When CEA is added in the Ru(NH3)63+/Apt@GO solution, DNA aptamer recognizes and binds to CEA and Ru(NH3)63+releases to the solution, which can be enriched and detected by VMSF/ITO electrodes. Based on this mechanism, CEA can be an electrochemical detection ranging from 60 fg/mL to 100 ng/mL with a limit of detection (LOD) of 14 fg/mL. Detection of CEA in human serum is also realized. The constructed homogeneous detection system does not require the fixation of a recognitive aptamer on the electrode surface or magnetic separation before detection, demonstrating potential applications in rapid, convenient and sensitive electrochemical sensing of tumor biomarkers.