Abstract: Native mass spectrometry (native MS) has emerged as an important analytical tool for the investigation of structures, compositions and intermolecular interactions of macromolecular assemblies, protein-drug complexes and nucleic acid-ligand complexes, directly from buffers that maintain their naturally folded structures and natively bound states. The non-volatile salts in biochemical buffers are essential for providing the sufficient ionic strength and appropriate pH, which are needed to maintain the native folded states of proteins and nucleic acids, as well as their binding states with various other molecules through the weak noncovalent interactions. Benefit from its unique matrix tolerance, a nanoscale capillary (nano-capillary) is capable of directly producing ions of proteins, nucleic acids and their ligand-bound complexes from physiological or near physiological buffers, which has greatly promoted the progress in native MS. In this review, the fabrication and characterization of a nano-capillary, the unique ionization behaviors caused by the ultrasmall size as well as its inner surface property of a nano-capillary were reviewed. The applications of native MS that employed a nano-capillary emerged during the last decade were also summarized.