Two-dimensional molecular junctions (MJs) are mostly developed by sandwiching molecules between two metal electrodes. Charge transport in molecular junctions is not only determined by the difference between work function of electrodes and HOMO/LUMO of the molecule (≈ energy offset, ε), but also on molecule–electrode electronic coupling strengths (Γ g). Detailed knowledge of molecule–electrode coupling could reveal its effect on electron transport efficiency. We have examined the modulation of electronic conductance (G) across bio-molecule/protein-based MJs, where electronic coupling strengths were altered via applied mechanical forces on molecules with conducting-AFM probe. We have utilized numerical tunneling transport models which are developed for MJs and calculated G, ε, Γ g from experimentally obtained current–voltage data. We conclude that the modulation in electronic transport in bio-MJs under applied forces originates from the alteration of Γ g, which further incites the alteration of physical structure and variation of electrostatics environment around the chromophore of the protein.