Herein we report a simple and rapid approach to fabricate a vertically-ordered mesoporous silica-electrochemically reduced graphene oxide nanocomposite film (VMSF/ErGO) on an indium tin oxide (ITO) electrode surface by the electrochemically-assisted self-assembly (EASA) method, which is capable of greatly promoting the electroanalytical performance of rutin compared to the previously reported VMSF modified ITO electrode and, meanwhile, displaying excellent anti-fouling and anti-interference ability in pharmaceutical formulations and human serum. Due to the excellent electrocatalytic activity of ErGO and the synergistic enrichment effects from the π-π interaction of ErGO and the hydrogen bond effect from the VMSF, the present VMSF/ErGO/ITO sensor is able to detect rutin with a wide linear range, a high sensitivity and a low limit of detection. Moreover, the VMSF/ErGO/ITO electrode could retain the high sensing performance with high transmittance upon tailoring the concentration of the modified graphene oxide (GO), which provides a simple approach for modifying transparent ITO electrodes and could be potentially used for optoelectronic devices.