Copper ions play an important role in various biochemical and physiological processes. Herein, for the first time, we report and investigate the electrochemical characteristics of polyurethane (PU)-modified electrodes to identify their potential use in sensing of copper ions. Using ferricyanide as the redox probe, PU alone exhibited weak electrochemical signals due to its low electrical conductivity. Therefore, new series of nanocomposites of PU with nano-structured materials such as silver, gold, copper, platinum and magnetic nanoparticles, in addition to the graphene oxide, multi-walled carbon nanotubes and manganese dioxide nanowires were developed, and their electrochemical performances were evaluated as electrode modifiers. The voltammetric signals showed that the electrode modified with PU-doped platinum nanoparticles (PU/Pt NPs) has depicted the highest voltammetric response with well-defined redox peaks. Hence, a full electrochemical assay was optimized and a linear range of copper ions from 100 ng/ml to 1000 ng/ml was achieved, while the calculated values of the limit of detection (LOD) as well limit of quantification (LOQ) were 16.72 ng/ml and 50.66 ng/ml, respectively. Eventually, copper ions were determined and validated in digested serum, urine, and acidified tap water samples, and the atomic force microscopy was used as the reference method for the validation. The developed PU-modified electrodes are cost-effective and depicted high environmental benefits since they allow the accurate detection of copper ions with no prior complicated treatments. Besides, the proposed method found to be highly sensitive and selective, and could be used successfully to determine the trace amounts of copper ions in real biological and environmental samples.