Abstract We employ Nafion‐mixed carbon dots (CDs) and low‐cost screen‐printed carbon electrodes (SPCEs) as foundation matrices for the fabrication of electrochemical biosensors. The NH 2 and COOH functional groups present on the SPCE surface after Nafion/CDs deposition allow for pyrophosphate ions (PPis) collection. Using Fe(CN) 6 3− as the electrochemical mediator, the SPCE‐Nafion/CDs are applied to the detection of aqueous PPi by square wave voltammetry. Between 50 and 1 μM, a linear connection is established between the square wave voltammetry current and the PPi concentration. The limit of detection is determined to be 1.01 μM, and recoveries of 113% (±1.9%) and 108% (±3.9%) are achieved for human urine samples spiked with 6 and 3 μM of PPi, respectively. Furthermore, this PPi assay is suitable for the usage of complicated urine matrices without the inclusion of heavy metals. We anticipate that this unique approach will be beneficial for PPi level monitoring in urine during therapeutic treatments of illnesses and malignancies.