An electrochemical sensor for doxycycline hyclate (DC) detection with high sensitivity and good selectivity is reported. The sensor was fabricated by electro-polymerization of molecularly imprinted polymers (MIPs) in the presence of DC onto multi-walled carbon nanotubes modified glassy carbon electrode (MWCNTs/GCE). The MWCNTs can significantly increase the current response of the sensor, leading to enhanced sensitivity. The MIPs provide selective recognition sites for DC detection. The experimental parameters, such as the polymer monomer concentration, supporting electrolyte pH, the time for electro-polymerization and the incubation time of the sensor with DC were optimized. Under optimized experimental conditions, the sensor displayed a linear range of 0.05μmol/L–0.5μmol/L towards DC detection, with the detection limit of 1.3 × 10-2μmol/L. The sensor was successfully applied for recovery test of DC in human serum samples.