Proteins are the main proton mediators in various biological proton circuits. Using proteins for the formation of long-range proton conductors is offering a bioinspired approach for proton conductive polymers. One of the main challenges in the field of proton conductors is to explore the local environment within the polymers, along with deciphering the conduction mechanism. Here, we show that the protonic conductivity across a protein-based biopolymer can be hindered using straightforward chemical modifications, targeting carboxylate- or amine-terminated residues of the protein, as well as exploring the effect of surface hydrophobicity on proton conduction. We further use the natural tryptophan residue as a local fluorescent probe for the inner local hydration state of the protein surface and its tendency to form hydrogen bonds with nearby water molecules, along with the dynamicity of the process. Our electrical and spectroscopic measurements of the different chemically-modified protein materials as well as the material at different water–aprotic solvent mixtures result in our fundamental understanding of the proton mediators within the material and gaining important insights on the proton conduction mechanism. Our biopolymer can be used as an attractive platform for the study of bio-related protonic circuits as well as a proton conducting biopolymer for various applications, such as protonic transistors, ionic transducers and fuel cells.