A highly sensitive electrochemical biosensor for the detection of trace amounts of promethazine has been designed. Double stranded (ds)DNA molecules are immobilized onto a pretreated glassy carbon electrode (GCE(ox)) surface. The voltammetric behaviors of promethazine on DNA-modified electrode were explored by means of cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The promethazine gave rise to a pair of well-defined peaks, which appeared at E(pc) = 52 mV and E(pa) = 96 mV (vs. Ag/AgCl) in 0.10 M acetate buffer (pH 5.0). The peak current was linearly enhanced with increasing the concentration of promethazine. The calibration was linear for promethazine over the range of 4.7 x 10(-10) to 9.3 x 10(-9) M with a correlation coefficient of 0.999. The limit of detection (LODs) was 3.0 x 10(-10) M (S/N = 3). The modified electrode was applied to determine promethazine in human blood samples with satisfactory results.