Haemoglobin (Hb) modified electrochemical devices have been prepared by Hb encapsulation in silica sol–gel films (SiO2), which were generated by electro-assisted deposition onto pyrolitic graphite electrodes (PGEs). The stability and electrocatalytic activity of Hb entrapped into SiO2 network was substantially enhanced in the presence of cationic surfactant (CTAB) and Au nanoparticles (Au-NPs). The composition of sol–gel synthesis medium, i.e., molar ratio of silica precursor to water, contents of Hb, CTAB and Au-NPs, as well as the conditions of electrogeneration had a great influence on the electrocatalytic activity of Hb on PGE surface. The electrochemical response of the PGE modified with the composite SiO2–Hb–CTAB–Au-NPs film was found to vary linearly with the concentration of dissolved oxygen in solution and this was exploited to determine this analyte in the tap water with detection limit 0.12 mg L−1. The electrocatalytic current of dissolved oxygen was also found to decrease in the presence of the antivirus drug––amino derivative of adamantane (rimantadine)––which opens the way to the determination of this drug with detection limit 0.3 mg L−1 using PGE modified with SiO2–Hb–CTAB–Au-NPs nanocomposite film.