A facile electrochemical sensor based on Ruthenium-Phosphate (Ru-Pi) was developed by electrodeposition of Ru-Pi on Polypyrrole (PPy) modified carbon fiber paper (CFP) electrode. Phosphate buffer solutions of neutral pH containing RuCl3 was used for voltammetric deposition of Ru-Pi on PPy/CFP electrode. The modified electrodes were characterized by High resolution transmission electron microscopy (HRTEM), High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), Field emission scanning electron microscopy (FESEM) with energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and electroanalytical techniques. A significant amount of phosphate (Pi) and Ru centers in higher oxidation states were present on Ru-Pi film deposited on PPy/CFP substrate. Pi plays a significant role during catalyst deposition and in its activity toward determination of Cholesterol. DPV studies demonstrated that under optimum conditions, the developed sensor has displayed a wide linear dynamic range between 0.16 nM and 20.0 nM with a superior detection limit of 0.54 x 10(-10) M. The proposed method was effectively applied in the nonenzymatic determination of cholesterol at an ultralow level in human blood serum samples. The method displayed high selectivity toward cholesterol in the presence of other interfering substances. (c) 2019 The Electrochemical Society.