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 air-water 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 potential-area isotherms, Brewster angle microscopy, and polarization modulation infrared reflection-absorption 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 air-water 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 air-water interface because of the formation of aggregates. The interfacial films were transferred to solid supports as Langmuir-Blodgett 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 UV-vis 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.