The immobilization of biomolecules in films with a controlled architecture permits the access of information on the molecular interactions, not only between film components, but also between the film and substances in the external environment. In this study, we investigated the immobilization of the phospholipase A2 from snake venoms (4-nitro-3-(octanoyloxy)benzoic acid, OBZ) in solid supports as a Langmuir-Blodgett (LB) film, followed by incorporation of carbon nanotubes (CNTs). The hybrid film was characterized by infrared spectroscopy and the interactions with its catalytic substrate were investigated. The presence of CNTs leads to a structure with an adequate environment to preserve the enzyme properties, leading to an optimum catalytic activity. This enhanced architecture was exploited in terms of vibrational spectroscopy, which indicated changes in the secondary structure of the enzyme upon contact with the catalytic substrate. © 2012 American Chemical Society.