Modification of carbon and gold electrodes by a biocomposite film based on silica, hemoglobin (Hb), and glucose oxidase (GOx) has been performed by electro-assisted deposition using an electrogenerated catalyst. Proteins immobilized in the film retain catalytic activity for three weeks. The stability of the analytical signal of a modified electrode significantly increases on the addition of Au nanoparticles of the diameter 20 nm and cetyltrimethylammonium bromide (20 CMC) to the sol. A gold electrode modified with a biocomposite SiO2–Hb–GOx–Au film has been used for the voltammetric determination of glucose in model solutions and porcine blood serum. The calibration curve is linear in the range 0.015–0.20 mM; the limit of detection is 0.008 mM of glucose. The developed method of modification offers promise for the preparation of sensing elements of two-enzyme amperometric biosensors.