An electrochromatographic microchip with carboxyl-group-derivatized mono-disperse silica packing was prepared from the corresponding colloidal silica solution by utilizing capillary action and self-assembly behavior. The silica beads in water were primed by the capillary action toward the ends of cross-patterned microchannel on a cyclic olefinic copolymer (COC) substrate. Slow evaporation of water at the front of packing promoted the self-assembled packing of the beads. After thermally binding a cover plate on the chip substrate, reservoirs for sample solutions were fabricated at the ends of the microchannel. The packing at the entrances of the microchannel was silver coated to fix utilizing an electroless silver-plating technique to prevent the erosion of the packed structure caused by the sudden switching of a high voltage DC power source. The electrochromatographic behavior of the microchip was explored and compared to that of the microchip with bare silica packing in basic borate buffer. Electrophoretic migration of Rhodamine B was dominant in the microchip with the carboxyl-derivatized silica packing that resulted in a migration approximated twice as fast, while the reversible adsorption was dominant in the bare silica-packed microchip. Not only the faster migration rates of the negatively charged FITC-derivatives of amino acids but also the different migration due to the charge interaction at the packing surface were observed. The electrochromatographic characteristics were studied in detail and compared with those of the bare silica packed microchip in terms of the packing material, the separation potential, pH of the running buffer, and also the separation channel length.