Creatinine is a preferred biomarker for the renal dysfunction, and its selective determination in various biological fluids is useful for the evaluation of renal, muscular and thyroid dysfunctions. Development of a selective and sensitive non-enzymatic electrochemical sensor having a linear response and good stability over a wide range of creatinine concentration is challenging. In this study, a polymeric dye-based metal-carbon nanofiber nanocomposite was successfully fabricated as the electrode of a non-enzymatic electrochemical sensor for creatinine in human biological fluids. Using cyclic voltametry technique, the dendritic poly methylene blue (PMB) nanofibers were electrochemically grown on the surface of the Cu-doped carbon electrode. The PMB molecule acted as a recognition element for creatinine. In virtue of the structural merits and synergistic contribution of the polymer-metal-carbon nanofiber nanocomposites in enhancing the electrocatalytic activity towards creatinine, the fabricated electrode exhibited good sensing performances including a high sensitivity of 0.133 μA ng mL−1, a linear response over 0.5–900 ng mL−1 conentration range and the detection limit of 0.2 ng mL−1 (with a signal-to-noise ratio of 3:1), as well as good selectivity. The proposed electrochemical sensor can be used for the real time detection of creatinine in biological fluids.