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For the first time, sulfanilamide (SFD) was determined in otologic solution, human urine and serum by electroanalytical techniques on glassy carbon electrode (GCE). The cyclic voltammetry (CV) experiments showed an irreversible oxidation peak at +1.06 V in 0.1 mol/L BRBS (pH = 2.0) at 50 mV/s. Different vol-tammetric scan rates (from 10 to 250 mV/s) suggested that the oxidation of SFD on the GCE was a diffusion-controlled process. Square-wave voltammetry (SWV) method under optimized conditions showed a linear response to SFD from 5.0 to 74.7μmol/L (R = 0.999) with detection and quantification limits of 0.92 and 3.10μmol/L, respectively. The developed SWV method showed better results for detection limit and linear range than the chronoamperometry method. It has been successfully applied to determine SFD concentration in pharmaceutical formulation, human urine and serum samples with recovery close to 100%.