The structure of enzymes must be conserved when incorporated in nanoelectronic devices because their activity determines the function of the device as sensors. Among the systems that can retain their conformational structures, Langmuir-Blodgett (LB) films can be useful to exploit the construction of bioelectronic devices organized at the molecular level because biological and polymeric materials can be coupled as ultrathin films for biosensors and actuators. In this paper, we immobilized a β-galactosidase enzyme in the LB films of stearic acid and the conjugated polymer poly[(9,9-dioctylfluorene)- co-thiophene]. After the characterization of the floating films using tensiometry, vibrational spectroscopy, and Brewster angle microscopy, they were transferred to solid supports as LB films, and the catalytic activity of the enzyme could be preserved as analyzed using UV-vis spectroscopy. We noted that the presence of a supramolecular structure formed in the LB films not only conserved the enzyme activity but also exhibited regular and distinctive output signals in all molecular architectures employed in this work. These results are related to the synergism between the compounds on the active layer associated with a surface morphology that facilitated the analyte diffusion because of an adequate molecular accommodation of all components. This work then demonstrates the viability of employing LB films composed of lipids, enzymes, and synthetic polymers as devices for biosensing applications.