A novel Au-Fe3O4 nanoparticles-based capillary column was fabricated by magnetic approach for open-tubular capillary electrochromatography (OT-CEC). The bifunctional dumbbell-like Janus Au-Fe3O4 nanoparticles (Au-Fe3O4 NPs) were prepared through a hydrothermal synthesis strategy, and the morphology was characterized by transmission electron microscopy (TEM). Multilayers Au-Fe3O4 NPs were easily coated onto the inner surface of silica capillary by an external magnetic field to generate an Au-Fe3O4 NPs-based column. Compared with a bare capillary, the modified surface exhibited more stable and suppressed electroosmotic mobility. The column showed good separation efficiency for neutral analytes in the OT-CEC separation mode, with theoretical plate numbers of up to 79705 per meter for naphthalene. The successful separation of dihydroxy benzene isomers and proteins demonstrated that the column exhibits a reasonable separation performance. The reproducibility of the Au-Fe3O4 NPs capillary was studied, with relative standard deviations (RSD) for day-to-day and column-to-column less than 1 and 1.75%, respectively.