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The preparative techniques of molecular multilayer films via the alternate adsorption of polyelectrolytes and biomolecules is one of the focal subjects in biometerials sciences due to the potential application for the construction of bioreactors, biomedical devices, and biosensors. Requirements for their sensitivity and selectivity force to look for new polymers with unusual architecture compared to conventional linear polyelectrolytes. The increased adsorption and improved fixation of biomaterial is expected due to unique properties of such polymers (non-linear charge distribution, different film morphology, mechanical properties, etc.) Herein the series of star-shaped poly(2-(N,N-dimethylamino)ethyl methacrylate), PDMAEMA, with 6, 9, and 17 arms completely quaternized with methyl iodide (PDMAEMAQ170)6, (PDMAEMAQ170)9, (PDMAEMAQ180)17, as well as the series of star-shaped poly(acrylic acid), PAA, with 5, 8, and 21 arms (PAA90)5, (PAA100)8, and (PAA100)21 were used as active building blocks for the incorporation of polyphenol oxidase and choline oxidase into self-assembled films deposited on graphite for the formation of biosensing surfaces for phenol and choline detection. It was found that spatial organization of the polyelectrolyte has significant influence on the properties of enzymes incorporated in self-assembled films. The comparison between the activities of enzymes incorporated into the films constructed from star polyelectrolytes and into the films made from linear arm analogues taken in equivalent compositions demonstrates pronounced increase in polyphenol oxidase and choline oxidase activities. Furthermore, the effect is getting to be greater with increasing the number of arms in polyelectrolytes used. The electrochemical activity of incorporated enzymes, their kinetic parameters (KM, VM) and the stability of self-organized films were studied amperometrically. Data are in well agreement with surface topography characteristics obtained by AFM.