Binary palladium–bismuth nanocatalysts supported on functionalized multi-walled carbon nanotubes (Pd–Bi/C) are synthesized using a one-pot polyol method. The prepared Pd–Bi/C catalysts have a metal particle range from 5.25 to 12.98 nm and are investigated for alkaline electrocatalytic glucose oxidation reaction (GOR). The physical properties of the catalysts are characterized by X-ray diffraction (XRD), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The electrochemical activities are determined by cyclic voltammetry (CV), linear sweep voltammetry (LSV), Tafel analysis and chronoamperomtry (CA) for comparing the electrochemical active surface area (ECSA), GOR onset potential, GOR peak current density, Tafel slope, poisoning rate and cycling stability of the Pd–Bi/C catalysts. It is found that Pd–Bi/C (1:0.14) can significantly enhance the electrocatalytic activity on GOR about 40% times higher than Pd/C and as well as has a 3.7-fold lower poisoning rate. The in-use stability of Pd–Bi/C (1:0.14) is also remarkably improved, according to the results of the 200 cycling CV test. The effects of the operating temperature and the concentration of glucose and NaOH electrolyte on Pd–Bi/C (1:0.14) are further studied in this work. The highest Pd–Bi/C catalyzed GOR current density of 29.5 mA cm−2 is attained in alkaline medium.