Metal–organic frameworks (MOFs) have a wide range of applications in separation and analytical sciences due to their unique structures. MIL-100(Fe) was immobilized on the inner wall of the capillary column based on the immobilized cysteine (Cys)-triggered in situ growth (ICISG) strategy. By applying this column to capillary electrophoresis, a novel enantioselective separation system based on MIL-100(Fe) nanomaterial was established. By utilizing lactobionic acid (LA) as the chiral selector and optimizing the experimental conditions such as buffer pH, LA concentration and the methanol addition ratio, the CEC system demonstrated a significantly enhanced enantioseparation ability for six basic drugs. The modified capillary column was characterized by scanning electron microscope (SEM), energy-dispersive X-ray spectroscope (EDS) and X-ray diffractometer. The experimental results showed that MIL-100(Fe) was successfully grown on the inner wall of the capillary column. In the present study, the repeatability of the coated column was investigated and the relative standard deviations (RSDs) of the resolution and the migration time for intra-day, inter-day and inter-column were within 8%, proving the good repeatability of the coated column.