Electrochemical techniques with excellent merits of rapid response and simple operation have attracted a lot of interest in the detection of biomolecules. Tyrosine (Ty) is a kind of essential amino acids in human and herbivores bodies. Dicyclomine (DI) is an antispasmodic and anticholinergic drug, a medication that reduces the effect of acetylcholine on smooth muscles. Altering the concentration of these species is cause to several diseases, so their determination in much essential. In the present study a glassy carbon electrode, modified with nanocomposite of gold nanoparticles/multiwalled carbon nanotubes (GNPs/MWCNTs/GCE) was used for the simultaneous determination of Ty and DI. The results showed that synergetic effects of GNPs and MWCNTs highly improved electrochemical response and sensitivity of the sensor. The electrochemical oxidation of Ty and DI was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Also, scanning electron microscopy (SEM) was used to evaluate microstructure of electrochemical sensor. The effect of various experimental parameters including pH and scan rate on the voltammetric response of Ty and DI were investigated. Under the optimal conditions linear response was observed in range of 4.0–130.0 µmol L 1 and 5.0– 120.0 µmol L 1 for Ty and DI, respectively. The lower detection limits were found to be 0.9 µmol L 1 and 0.55 µmol L 1 for Ty and DI, respectively. The investigated method showed good stability, reproducibility and repeatability. The proposed sensor was successfully applied to the simultaneous determination of Ty and DI in real samples.