Antibiotics are extensively used in veterinary medicine for the treatment of bacterial infections. However, their uncontrolled use can cause infiltration into milk and meat, which can cause antimicrobial resistance. Therefore, the development of sensitive and selective methods for the detection of antibiotics is an urgent need. Conventional methods such as HPLC or ELISA can detect antibiotics with high sensitivity; however, they require experienced personnel, expensive antibodies, and expensive instruments. Biosensor technology is an alternative to conventional analytical methods. A biosensor is composed of sensing layers with immobilized receptors and a transducer that convert chemical signals into measured electrical, optical, or acoustic values. DNA aptamers are relatively novel receptors that are also known as chemical antibodies. In contrast with antibodies, they are more stable and can be immobilized on various surfaces. We developed an electrochemical aptasensor based on a nanocomposite of reduced graphene oxide (rGO) and multiwalled carbon nanotubes (MWCNTs) enriched with carboxylic groups for sensitive detection of oxytetracycline (OTC). The amino-modified DNA aptamers were covalently immobilized on rGO-MWCNTs layers drop-casted on a glassy carbon electrode (GCE). Differential pulse voltammetry (DPV) in the presence of 5 mM [Fe(CN)6]^3-/4- was used for OTC detection. The limit of detection (LOD = 0.46 ng/mL) was much lower than the maximum residue limit (MRL) established for OTC by the EU (100 ng/mL). The selectivity of the sensor was demonstrated by using kanamycin, penicillin, chloramphenicol, and tetracycline antibiotics. The aptasensor was validated in 3.5 % fat milk with successful recovery.