MicroRNAs (miRNAs) are clinical biomarkers for various human diseases, including cancer. They have been found in liquid biopsy samples, including various bodily fluids. They often play an important role in the early diagnosis and prognosis of cancer, and the development of simple and effective analytical methods would be of pivotal importance for the entire community. The determination of these targets may be affected by the different physicochemical parameters of the specimen of interest. In this work, an electrochemical detection platform for miRNA based on a screen-printed gold electrode was developed. In the present study, miRNA-21 was selected as a model sequence, due to its role in prostate, breast, colon, pancreatic, and liver cancers. A DNA sequence modified with methylene blue (MB) was covalently bound to the electrochemical strip and used to detect the selected target miRNA-21. After optimization of selected parameters in standard solutions, including the study of the effect of pH, the presence of interferent species, and NaCl salt concentration in the background, the application of square-wave voltammetry (SWV) technique allowed the detection of miRNA-21 down to a limit in the order of 2 nM. The developed device was then applied to several urine samples. In this case too, the device showed high selectivity in the presence of the complex matrix, satisfactory repeatability, and a limit of detection in the order of magnitude of nM, similarly as what observed in standard solutions.