Vitamin B-12 is very important for human metabolism and its deficiency can cause anemia and the production of large red blood cells. An increased concentration of methylmalonic acid (MMA) is detected much before the transformation of blood cells, which thereby is an early indicator for mild or serious Vitamin B-12 deficiency. A simple electrochemical sensor based on Palladium-Gold (PdAu) was developed by electrodeposition of PdAu nanoparticles on Polypyrrole (PPy) modified carbon fiber paper (CFP) electrode. The modified electrodes were characterized by High resolution transmission electron microscopy (HRTEM), Field emission scanning electron microscopy (FESEM) with energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and electroanalytical techniques. Differential Pulse Voltammetric (DPV) studies have established that under optimum conditions, the developed sensor exhibits a broad linear dynamic range (4.01 pM - 52.5 nM) with a very low detection limit (1.32 pM). The proposed method was effectively applied in the non-enzymatic determination of MMA at an ultralow level in human blood serum and urine samples. The method displayed high selectivity toward MMA in the presence of other interfering substances.