Developing ordered mesoporous silica-based membranes with highly sensitive detection performance is of great significance for direct electrochemical detection in biological fluids against surface biofouling. Herein, we describe a facile one-step approach for the integration of a vertically-ordered mesoporous silica-nanochannel film (VMSF) with electrochemically reduced graphene oxide (ErGO) by an electrochemically assisted self-assembly (EASA) method. Electrochemical reduction of GO and growth of the VMSF simultaneously occur in a simple one-step process, forming a VMSF/ErGO layer on the glassy carbon electrode (GCE). Arising from the oxygen-containing groups, hydrophobic π-conjugated structure, and the two-dimensional planar structure of ErGO, the VMSF could be stably grown on the GCE surface and it further acted as a protective layer to prevent the internal ErGO electroactive layer from falling off the electrode surface over prolonged use. In comparison with a bare GCE, the present VMSF/ErGO/GCE sensor exhibits an excellent response to doxorubicin with a wide linear range (1 nM−20 μM), a high sensitivity (7.815 μA μM⁻¹) and a low limit of detection (0.77 nM), due to the cooperative signal amplification originating from the electrocatalytic activity and π–π interaction of ErGO and electrostatic preconcentration effect of the VMSF. Furthermore, considering the outstanding anti-fouling and anti-interference ability of the VMSF, direct determination and long-term monitoring of doxorubicin in human whole blood was successfully achieved by the sensor.