In order to study the post-polymerization functionalization of a poly(N-substituted pyrrole) film, the β-ferrocene ethylamine used as redox probe has been immobilized via a chemical coupling on the surface of a preformed polypyrrole film bearing activated ester groups. From the electrochemical response of the anchored ferrocene units, the post-polymerization functionalization efficiency has been evaluated to be 20–22%. This level is the same whatever the length of spacer arm (2, 5 and 12 carbons) linking the activated ester groups to the polypyrrole backbone and the hydrophilic and hydrophobic nature of this polymer. Using a similar strategy based on this chemical grafting, we have developed a new approach to the immobilization of biotin entities onto the ester activated-polypyrrole film surface. From fluorescence measurements, it appears that the anchoring of streptavidine bearing a fluorophore (streptavidin-R-phycoerythrin, SAPE) on this biotinylated polypyrrole film gives a fluorescence intensity similar to the one achieved from the immobilization of SAPE onto a film electrosynthesized from pyrrole monomers bearing biotin units. Thus, this result shows the efficiency of this new functionalization method for attaching biotinylated entities.