A novel molecularly imprinted electrochemical sensor for sensitive determination of oxycodone was developed. Gold nanoparticles (G) and multi-walled carbon nanotubes (MWCNTs) were used for enhancement of electronic transmission and sensitivity. The thin film of molecularly imprinted sol-gel polymers with specific binding sites for oxycodone was cast on modified pencil graphite electrode by electrochemical deposition method. The resulting composite was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetric techniques (CV). The interaction between the imprinted sensor and target molecule was observed by measuring the current response of the oxidation–reduction of the K 3 [Fe(CN) 6 ] as a probe, on the electrode. Under the optimal experimental conditions, the response peak current was linear to the concentration of oxycodone in the range from 0.001 to 0.08 µM and 0.08 to 5 µM the detection limit was 0.7 nM and recovery percent was more than 94% and RSD was 4.7%. This imprinted sensor was successfully applied to the determination of oxycodone in human urine samples. These results have revealed that the imprinted sensor fulfilled the selectivity, sensitivity,speed and simplicity requirements for oxycodone detection and provided possibilities of clinical application in physiological fluids.