Pure cardiolipin deposit onto electrodes is optimized and shown to yield an efficient supported lipid film for promoting cytochrome-c immobilization and electroactivity. Cyclic voltammetry and electrochemical impedance spectroscopy measurements in an aqueous electrolyte with potassium ferri- and ferrocyanide as a redox probe evidence that an optimized pure cardiolipin film is reached for a 7 μg cm− 2 deposit onto glassy carbon electrode. At this optimized surface concentration the pure cardiolipin deposit yields the most compact and less permeable supported lipid film on electrode surface. The thickness and the organization of the pure cardiolipin films were analyzed by atomic force microscopy (AFM) measurements. AFM imaging in aqueous buffer shows that the lipid deposit onto the surface forms a thick deposit of approximately 30 ± 10 nm of height with 4 nm average roughness and includes defects. Cytochrome-c electroactivity was studied with the redox protein either in solution or immobilized onto the modified electrode. First, the optimized amount of pure cardiolipin was deposited onto glassy carbon electrodes to study the stable and electrochemically quasi-reversible redox system of cytochrome-c in solution. Then, the potential cycling of a pure cardiolipin-modified glassy carbon electrode in a cytochrome-c solution led to the immobilization of the protein in its native state keeping intact its electrochemical properties, and with a surface coverage of 8 pmol cm− 2 corresponding to 50% of a monolayer.