The preconcentration, separation, and electrochemical detection of a series of tetracycline (TC) antibiotics in a microfluidic channel were preformed. The electrophoretic experimental parameters to analyze TC, oxytetracycline, chlortetracycline, and doxycycline antibiotics were investigated with a cellulose-dsDNA-modified carbon paste electrode. Modification of the electrode improved detection performance by enhancing the signal-to-noise characteristics without surface fouling of the electrode. Field-amplified sample stacking and field-amplified sample injection techniques were employed for on-chip preconcentration of the TC series. The sensitivity of the method was improved 10 900-fold when compared with conventional MEKC-electrochemical detection analysis. The overall recoveries for TC, oxytetracycline, chlortetracycline, and doxycycline were 87, 89, 87, and 81%, respectively, with 6.0% RSD. The limits of detection for the series were estimated between 1.5 and 4.3 nM. Applicability of the method to beef samples was successfully demonstrated.