A simple and sensitive method is proposed for the electrochemical determination of enrofloxacin (ENRO) and its primary metabolite ciprofloxacin (CIPRO) at a multiwall carbon nanotubes/glassy carbon electrode (MWCNT/GCE) using a least-squares support vector machine (LS-SVM) and linear sweep voltammetry. Simultaneous determination of ENRO and CIPRO at bare glassy carbon is associated with certain difficulties due to voltammogram overlapping and their low sensitivity. The resolution of the mixture was carried out using LS-SVM as a multivariate calibration method. Under the optimum conditions at pH 7.0, the linear sweep currents increased linearly with ENRO and CIPRO concentrations in ranges of 2.0 − 780.0 μmol L⁻¹ (0.7 − 280.3 μg mL⁻¹) and 3.0 − 1200 μmol L⁻¹ (1.0 − 397.7 μg mL⁻¹), respectively. The detection limits for ENRO and CIPRO were 0.5 and 0.9 μmol L⁻¹, respectively. The proposed method was applied to simultaneously determine both compounds in human urine, plasma and in pharmaceutical samples.