A novel glucose biosensor was developed based on a 4-amino thiophenol/Au nanoparticle/glucose oxidase (GOD)–horseradish peroxidase (HRP)/6-mercapto-1-hexanol-11-mercaptoundecanoic acid/Au electrode. The modified electrode could be used to detect glucose based on the polymerization of aniline induced by HRP and H₂O₂ which was produced by the reduction of O₂ accompanied by the oxidation of glucose into gluconic acid via GOD. With the increase of glucose, more H₂O₂ molecules were produced and then a growing number of polyaniline (PANI) molecules were formed on the modified electrode surface, which resulted in the decrease of peak current of Fe(CN)₆^3−/4− because the formed PANI hindered the electron transfer of Fe(CN)₆^3−/4− toward the electrode surface. Based on the decrease of peak current of Fe(CN)₆^3−/4−, the glucose concentration could be determined accurately and selectively. The resulted biosensor exhibited a wide linear range from 16.5 μM to 10.0 mM, a high sensitivity of 41.78 μA mM⁻¹ cm⁻² and good selectivity. The glucose biosensor exhibits some advantages over the traditional GOD electrodes in terms of a close to zero applied potential. Moreover, the used electrode might be used as a H₂O₂ sensor due to its good electrocatalytic response for H₂O₂.