Kelvin probe force microscopy (KPFM) is a highly advanced technique offering notable surface sensitivity and high lateral resolution, ranging from micrometres to the sub-nanometre scale. This scanning probe technique effectively detects local electrical surface potential (ESP), influenced charge distribution, and work function differences, making it essential for studying biological and biochemical processes, from single molecules to complex cellular structures. By enabling nanometre-resolution analysis under simulated conditions, KPFM provides crucial insights into the physicochemical evolution, functionality, and structural organization of biomolecular systems. Recent advancements have significantly expanded KPFM's capabilities, revealing ESP characteristics in diverse biological entities, including single proteins, DNA strands, lipid films, fibrils, and complex neuronal structures. The technique also facilitates the study of biomolecular nanolayers on advanced nanomaterials like gold nanoparticles and carbon nanotubes, enhancing its role in bio-nanotechnology. Such versatility highlights KPFM's transformative potential in elucidating biomolecular interactions at unprecedented resolutions. This review critically analyses recent advancements, addresses ongoing challenges in measuring ESP in biological samples, and highlights emerging strategies to improve resolution and sensitivity. Additionally, KPFM's implications in diagnostics, biosensing, tissue engineering, therapeutics, drug screening, and Alzheimer's research are explored, establishing it as a powerful tool at the intersection of nanotechnology and biomedical innovation.