The invention discloses a preparation method of superficially-porous core-shell-structured silica gel microspheres and chromatographic separation application of the silica gel microspheres. The method comprises: accumulating H2SiF6-hydrolysed nanometer silica on the surface of spherical silica gel to obtain superficially-porous core-shell-structured silica spheres containing a certain pore diameter, and modifying the surface of the silica spheres with different functional groups. The obtained silica gel microspheres can be applied to macro-molecule compounds, such as protein and polypeptide, and separation analysis of various different small-molecule compounds. The method adopts cheap raw materials, is simple and controllable, is short in preparation time, and allows porous shells with different pore diameters and thicknesses to be prepared. The prepared stationary phase can be widely applied to separation analysis of macro-molecule compounds and fast separation analysis of small-molecule compounds.