A polyelectrolyte multilayer combining cytochrome c (cyt.c) and xanthine oxidase (XOD) is assembled on a gold electrode and investigated with respect to a signal chain formation from a xanthine oxidase substrate in solution. The multilayer assembly is prepared by means of an electrostatic self-assembly technique and consists of two parts each comprising one type of protein, which is responsible for a specific function. The outer part of the film contains XOD immobilized within poly(ethylenimin) layers and is responsible for selectivity towards hypoxanthine (HX) by its enzymatic conversion to uric acid. The inner layers contain cyt.c molecules embedded into a sulfonated polyaniline (PASA) matrix. The signal transfer mechanism within the assembly is proposed to be a mediated one. Thus, cyt.c plays a role of an internal transducer translating a HX concentration into an amperometric electrode response. Formation of the multilayer structure is confirmed by surface plasmon resonance (SPR), electrochemical experiments and UV-vis spectrophotometry. Influence of the multilayer composition on sensor performance is discussed. (c) 2007 Elsevier Ltd. All rights reserved.