In this study, the carbon nanotube supported gold, bismuth, and gold-bismuth (Au/MWCNT, Bi/MWCNT, and Au-Bi/ MWCNT) nanocatalysts were prepared with NaBH4 reduction method at varying molar atomic ratio for glucose electrooxidation (GAEO). The synthesized nanocatalysts at different Au: Bi atomic ratios are characterized via x-ray diffraction (XRD), transmission electron microscopy (TEM), and N-2 adsorption-desorption. For the performance of AuBi/MWCNT for GAEO, electrochemical measurements are performed by using different electrochemical techniques namely cyclic voltammetry (CV), linear sweep voltammetry (LSV), chronoamperometry (CA), and electrochemical impedance spectroscopy (EIS). Monometallic Au/MWCNT exhibits higher activity than Bi/MWCNT with 256.57 mA/mg (0.936 mA/cm(2)) current density. According to CV results, Au80Bi20/MWCNT nanocatalyst has the highest GAEO activity with the mass activity of 320.15 mA/mg (1.133 mA/cm(2)). For Au80Bi (20)/MWCNT, central composite design (CCD) is utilized for optimum conditions of the electrode preparation. Au80Bi20/MWCNT nanocatalysts are promising anode nanocatalysts for direct glucose fuel cells (DGFCs).