We demonstrated a highly sensitive double-fluorescent dye staining in microchip electrophoresis (ME) for analysis of milk proteins. The detection sensitivity of ME was very limited so far and needed improvement. Our staining method consisted of two steps. First, in sample preparation before electrophoresis, protein was covalently bound to an amine-reactive fluorescent dye, Cy5. Then, the Cy5-attached protein was denatured with SDS and was further stained, during electrophoresis, with Agilent fluorescent dye, which was non-covalently attached to hydrophobic regions of the SDS-protein complexes. This double-fluorescent staining enhanced fluorescent intensity and lowered the detection limit to 200 pg of protein. This provided higher sensitivity than silver- or SYPRO Ruby-staining methods, which have previously given the highest sensitivity in protein staining. In addition, we applied our staining method to analysis of milk proteins and achieved their successful detection, whereas it was difficult to analyze them by the unimproved method.