Noncovalent chemical interactions, including electrostatic interaction, hydrogen‐bonding, and hydrophobic interaction, constitute crucial driving forces in the bottom‐up assembly of multifunctional molecular complexes and nanomaterials. Electrostatic interaction in chemical environments is generally considered as non‐specific interaction, which poses notable challenges for control and direct measurement of electrostatic forces at the single‐molecule level. Here, we report a model system consisting of cytochrome c and carboxyl group‐containing molecule and use electrochemical AFM force spectroscopy to navigate the electrostatic forces of the system under various chemical conditions. Control and effects of solution pH, ion strength, and redox states of cytochrome c on electrostatic interactions are systematically studied. This work would provide a useful approach to investigate the intermolecular electrostatic interaction at the single‐molecule level.