Adsorption and enzyme activity of sucrose phosphorylase on lipid Langmuir and Langmuir-Blodgett films. The production of bioelectronic devices, including biosensors, can be conducted using enzymes immobilized in ultrathin solid films, for which preserving the enzymatic catalytic activity is crucial for optimal performance. In this sense, nanostructured films that allow for control over molecular architectures are of interest. In this paper, we investigate the adsorption of sucrose phosphorylase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid, which caused the surface pressure isotherms to expand. With polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), the amide bands from the enzyme could be identified, with the C-N and C=O dipole moments lying parallel to the air-water interface. Structuring of the enzyme into an α-helix was noted, and this structure was preserved when the mixed enzyme-phospholipid monolayer was transferred in the form of a Langmuir-Blodgett (LB) film. The latter was demonstrated with measurements of the catalytic activity of sucrose phosphorylase, which presented the highest enzyme activity for multilayer LB film. The approach presented in this study not only allows for optimized catalytic activity toward sucrose but also permits to explain why certain film architectures exhibit superior performance.