Scanning probe microscopy (SPM) techniques demonstrate one of the most promising tools to investigate the physical and chemical properties of materials at nanoscale and become the most common and important characterization tools in the field of nanotechnology. Among many SPM methods electrochemical scanning tunneling microscopy (EC-STM) technique is one technique that directly provides three-dimensional real-space images with in-situ interfacial electrochemical studies and allows locally measured properties of nanostructured materials at atomic resolution. Furthermore, EC-STM based studies provides information on solution covered areas of electrode surfaces, metal deposition, charge transfer, potential-dependent surface morphology, corrosion, semiconductors, and various applications such as protein conductance measurements in nanobioelectronics. Therefore, in this review, we summarize the electrochemical scanning tunneling microscopic investigation on protein based electrode structures and their applications towards novel bioelectronic devices along with recent developments in ECSTM techniques with future prospects in the field of nanobiotechnology.