In this study, we modified a poly(toluidine blue O) (TBO) film on a Au electrode by electropolymerization and investigated its electrical characteristics when used as a potentiometric biosensor. The fundamental properties of poly-TBO films such as immobilization density and thickness were clarified by atomic force microscopy (AFM) for different electropolymerization times. The poly-TBO film electrode with a spikelike surface, which was formed during electropolymerization, showed pH sensitivity (45.5 mV/pH) near the Nernstian response when used as a potentiometric sensor. This behavior is assumed to arise because the charge density at a spikelike film is higher than that at a smooth film. Thus, a platform based on a potentiometric biosensor with a poly-TBO film electrode can be used as a bioanalytical system to measure ionic behaviors that involve biomolecular recognition. This will enable the measurement of negatively charged sulfated glycosaminoglycan (sGAG), which is included in the extracellular matrix (ECM) generated from chondrocytes and is used as an important indicator in the development of articular cartilage in the field of regenerative medicine because of its electrostatic binding to positively charged TBO molecules.