Electromembrane extraction was used with high‐performance liquid chromatography for preconcentration and determination of ampicillin residues in cow milk. Ampicillin is transferred from an aqueous solution through a thin layer containing octan‐1‐ol, silver nanoparticles, and reduced graphene oxide which serves as a supported liquid membrane. Inside the fiber impregnated with supported liquid membrane mixture was filled 10 µL of an acceptor phase. Experimental parameters were optimized for extraction efficiency of ampicillin. Under the optimized conditions, the proposed method provided acceptable linear range (2–100 µg/L), satisfactory repeatability (RSD% < 7.1), low limit of detection (0.6 µg/L), and a high enrichment factor (295) corresponding to extraction recovery of 37%. Consequently, the proposed method was successfully applied for the determination of ampicillin residues in different cow milks.