A sensitive and reliable stripping voltammetry method was developed to determine the presence of Ceftiofur antibiotic drug. This method is based on the adsorptive accumulation of the drug at a hanging mercury drop electrode and then the initiation of a negative sweep that yielded well-defined cathodic peaks at 20.65 V (1 C) and 21.00 V (2 C) vs. Ag/AgCl reference electrode. To achieve high sensitivity, various experimental and instrumental variables were investigated such as supporting electrolyte, pH, accumulation time and potential, drug concentration, scan rate, convection rate, and working electrode area. The monitored adsorptive current of peak 1 C was directly proportional to the concentration of Ceftiofur; it shows a linear response in the range from 0.50 Â 10 28 to 8.00 Â 10 28 mol L 21 (correlation coefficient ¼ 0.998); and the limit of detection is 6.00 Â 10 210 mol L 21 at an accumulation time of 300 s. The applicability of this approach was illustrated by the determination of Ceftiofur in pharmaceutical preparations and bovine serum.