We focused on a rapid method for the assay of protein unbound indoxyl sulfate (IS) in urine samples by square-wave voltammetry. A sensor based on the electroactivity of commercial graphene screen-printed electrode (GR-SPE) for sensitive estimation of uremic toxin indoxyl sulfate was presented. The behaviors of uremic solute indoxyl sulfate on GR-SP electrodes were explored by cyclic and square-wave voltammetry. The outcomes displayed that the GR-SP electrode exposed perfect electroactivity to uremic toxin indoxyl sulfate and the oxidation of uremic solute IS is a multi-step and irreversible process. The process is pH dependent and takes place with the forging of a main oxidation product that is adsorbed on the GR-SP electrode surface. The calibration curve was achieved in the IS concentration range of 0.5-80 mu M and the limit of detection was calculated to be 0.064 mu M at a signal-to-noise ratio of 3. The use of the commercially disposable GR-SPE in human serum and urine sample assays obtained satisfactory results, which made it a promising alternative in routine sensing applications.