We discuss here real-time monitoring of the formation of biocatalytically active films of membrane-bound human cytochrome P450 3A4 expressed with its reductase in E. Coli (so called bactosomes) on a cysteamine self-assembled monolayer of gold infused quartz crystals. Amount of immobilized P450 3A4 bactosomes was 471±21 μg cm^–2 gold geometric area (N = 3 replicates). Scanning electron microscopic and impedance characterizations of the constructed P450 bactosomal films, electrocatalytic oxygen reduction activity, and voltage-driven (–0.6 V vs. Ag/AgCl, 1 M KCl) C-H functionalization of diclofenac to 4-hydroxydiclofenac typical of P450 3A4 catalysis are presented. The formation of 4-hydroxydiclofenac in the presence of added catalase (hydrogen peroxide scavenger) to the electrolysis solution suggested the reductase pathway of P450 3A4 catalysis followed in the designed bactosomal film, which is similar to the in vivo catalytic mechanism. This one-step construction of biocatalytically active films of cost-effective P450 bactosomes is quite useful for low cost, stereoselective, and NADPH-free drug metabolism assays, biosensing, and biocatalysis applications.