Nanopipettes, as a class of solid-state nanopores, have evolved into universal tools in biomedicine for the detection of biomarkers and different biological analytes. Nanopipette-based methods combine high sensitivity, selectivity, single-molecule resolution, and multifunctionality. The features have significantly expanded interest in their applications for the biomolecular detection, imaging, and molecular diagnostics of real samples. Moreover, the ease of manufacturing nanopipettes, coupled with their compatibility with fluorescence and electrochemical methods, makes them ideal for portable point-of-care diagnostic devices. This review summarized the latest progress in nanopipette-based nanopore technology for the detection of biomarkers, DNA, RNA, proteins, and peptides, in particular beta-amyloid or alpha-synuclein, emphasizing the impact of technology on molecular diagnostics. By addressing key challenges in single-molecule detection and expanding applications in diverse biological areas, nanopipettes are poised to play a transformative role in the future of personalized medicine.