Simultaneous determination of deferoxamine and vitamin C at the surface of a nanostructured -modified sensor is reported at this study. The electrode is designed based on a carbon paste electrode (CPE) modified with 1-ethyl-3-methylimidazolium chloride as an ionic liquid (IL) and CdO-nanoparticle/rGO (IL/CdO/rGO/CPE). The nanostructures are studied by various techniques containing X-ray diffraction (XRD), energy dispersive X-ray (EDX), field emission scanning electron microscopes (FESEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). The high degree of overlapping voltammograms is studied by multivariate curve resolution alternating least (MCR-ALS) algorithm. At first, a multivariate optimization is studied using response surface methodology (RSM) and central composite rotatable design (CCRD) model based on a five-level, three factor to optimize various parameters on DPV signal of the drugs. Then, second order data are built by changing in pulse heights of DPV. Results of MCR-ALS are used to data analysis that is obtained by electro-oxidation of deferoxamine and vitamin C. The strategy shows the current method is a potential way for analysis of the two important drugs in the presence of each other with high voltammetric overlapping in human serum and urine samples of patients with thalassemia major and minor. The voltammetric analyses of deferoxamine and vitamin C are performed in the concentration range of 5.0 × 10−8−900.0 × 10−2 and 6.0 × 10−8−900.0 × 10−2 M, and the detection limits are calculated 0.030 × 10−6 and 0.025 × 10−6 M, respectively.