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Electromembrane extraction coupled with high‐performance liquid chromatography (HPLC) and ultraviolet (UV) detection was developed for the determination of levamisole in some human biological fluids. Levamisole migrated from 4 mL of different acidized biological matrices, through a thin layer of 2‐nitrophenyl octyl ether containing 5% tris‐(2‐ethylhexyl) phosphate immobilized in the pores of a porous hollow fiber, into a 20‐μL acidic aqueous acceptor solution present inside the lumen of the fiber. The parameters influencing electromigration were investigated and optimized. Within 15 min of operation at 200 V, levamisole was extracted from different biological fluid samples with recoveries in the range of 59–65%, which corresponded to preconcentration factors in the range of 118–130. The calibration curves showed linearity in the range of 0.5–10, 0.2–10 and 0.1–10 μg/mL for plasma, urine and saliva …