In this work, the copolymer poly[(9,9-dioctylfluorene)-co-(3-hexylthiophene)] was employed as a matrix for immobilizing phytase, aiming at the detection of phytic acid. The copolymer was spread on the airwater interface forming Langmuir monolayers and phytase adsorbed from the aqueous subphase. The interactions between the copolymer and the enzyme components were investigated with surface pressure and surface potentialarea isotherms, Brewster angle microscopy, and polarization modulation infrared reflectionabsorption spectroscopy (PM-IRRAS). The enzyme could be incorporated in the monolayers from the aqueous subphase, expanding the copolymer films and maintaining its secondary structure. The polymeric films presented a morphological heterogeneous pattern at the airwater interface because of the ability of their chains to fold and entangle, causing inherent defects in the organization as well as unbalanced lateral distribution at the airwater interface because of the formation of aggregates. The interfacial films were transferred to solid supports as LangmuirBlodgett films and characterized by PM-IRRAS and scanning electronic microscopy, which showed not only the co-transfer of the enzyme but also the maintenance of their heterogeneous morphological pattern. The enzymatic activity of the blended film was analyzed by UVvis spectroscopy and allowed the estimation of the value of the Michaelis constant (13.08 mM), demonstrating the feasibility of the system to selectively detect phytic acid for biosensing purposes.