Different ratios of PtAu/C electrocatalysts were synthesized and assessed for their capability by glycerol electrooxidation. Electrocatalysts were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), cyclic voltammetry (CV), chronoamperometry, and direct glycerol/air fuel cell coupled with a ATR-FTIR setup. XRD of PtAu/C electrocatalysts showed the presence of Pt (fcc), Au (fcc), and PtAu (fcc) phases, and TEM images for PtAu/C electrocatalysts showed particle size between 5.4 and 5.8 nm. PtAu/C (50:50) presented the best result for glycerol electrooxidation by cyclic voltammetry and chronoamperommetry measurements in comparison with other electrocatalysts prepared. All PtAu/C showed better performance in comparison with Pt/C and Au/C. Moreover, the addition of gold to platinum favored glycerol by electronic effect and bifunctional mechanism. Through ATR-FTIR/direct alkaline glycerol/air cell in-situ study, it was possible to identify glycerate and tartronate as main products formed during the electrochemical glycerol oxidation.