Polypyrrole (Ppy) has been shown as a matrix for label-free electrochemical immunosensor based on electrochemical impedance spectroscopy (EIS) measurements. The immunosensing system model presented here was based on bovine leukemia virus (BLV) protein (gp51) entrapped within electrochemically-synthesized polypyrrole (Ppy/gp51). This Ppy/gp51 layer interacted with antibodies against gp51 (anti-gp51-Ab) that are present in significant concentration in the blood serum of BLV infected cattle. After this interaction protein complex (Ppy/gp51/anti-gp51-Ab) was formed. The horseradish peroxidase (HRP) labeled secondary antibodies (Ab) against anti-gp51-Ab were applied as agents interacting with Ppy/gp51/anti-gp51-Ab and forming the large protein complex (Ppy/gp51/anti-gp51-Ab/Ab). The EIS study was performed for electrodes modified with different Ppy layers described here and an optimal equivalent circuit was adopted for evaluation of EIS spectra, it was a major outcome of this study.