Selective detection of creatinine (Ctn) is crucial for the clinical diagnosis of early-stage kidney dysfunction. A non-enzymatic electrochemical method for a highly selective and sensitive detection of Ctn in blood serum and urine samples was developed using an electrochemically activated carbon-coated paper-based analytical device (ePAD*). During activation, the growth of stable carboxyl (C = O, COOH) groups on the edge planes of the carbon surface was observed. The prepared ePAD* was characterized using various microscopic, spectroscopic, and electrochemical techniques. The ePAD* was utilized as a medium exchanger, and the voltammetric response of Ctn detection in the linear concentration from 0.03 to 45 mM with a detection limit of 5.41 mu M was achieved. In addition, the selective detection of Ctn in the presence of potential interferences (i.e., uric acid, ascorbic acid, ammonia, and urea) was examined. The examination revealed a remarkable selectivity toward Ctn detection. Real-time detection of Ctn in blood serum and urine samples was performed. The results were correlated with the classical Jaffe <acute accent> colorimetric method. Our ePAD* exhibited a better electrochemical response. Thus, it is a highly potential diagnostic method for further development as a rapid and precise detection platform for point-of-care (POC) devices for examining patients with kidney disease.