The multilayer formation of two different sulfonated polyanilines with cytochrome c is presented and mechanistic aspects of the contributions of the polyelectrolytes' properties to the characteristics of the assemblies are discussed. These two modified polymers, PASA1 and PASA2 are chemically synthesized and differ in the grade of sulfonation, substitution, and the chain length of the polymer. The influence of these properties on the multilayer assembly with cytochrome c is studied in detail by Quartz Crystal Microbalance (QCM) technique and Cyclic Voltammetry (CV). It is shown that the multilayer formation is successful, however, the redox activity of polyanilines itself has to be taken into account. In the case of a strong redox activity (PASA2) voltammetric analysis allows the separation of redox processes addressed to the polyelectrolyte and cyt c. For multilayers with PASA1 as building block electroactivity can be predominantly attributed to cyt c ensuring a high amount of electroactive protein and a low probability for interfering redox reaction, making this system suitable for biosensor applications.