An MCE electrochemical enzyme immunoassay protocol for the determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was reported. Two antigens (Ag), CEA and AFP, were incubated simultaneously with an excess amount of horseradish peroxidase-labeled antibody (Ab*). The free Ab* and the Ab*-Ag complex produced in the solution were first separated through a postcolumn reaction and then traced by the enzyme substrate o-aminophenol. The 3-aminophenoxazine produced in enzyme reaction was detected with downstream amperometric detection. The separations were performed at a separation voltage of + 1.4 kV and were completed in less than 60 s. The better analytical performance and distinct miniaturization/portability for MCE at less assay time and sample volume consumption was achieved. The detection limit of CEA and AFP was calculated to be 0.25 and 0.13 ng/mL, respectively. Therefore, MCE could be used as a sensitive and new tool in separation science and offered considerable promise in biological sample analysis or quick clinical diagnosis.