The pH dependent redox potential of the oxidoreductase glucose oxidase (GOx) from Aspergillus niger, which is the most frequently applied enzyme in electrochemical glucose biosensors and biofuel cells, was measured between pH 4.5 and 8.5 using UV/vis spectroelectrochemistry. In the entire pH range under investigation, the flavin adenine dinucleotide cofactor of GOx changed directly from the oxidized quinone to the doubly reduced hydroquinone. No stable semiquinoid species could be detected if electrochemical equilibrium was reached. From the pH dependency of the GOx redox potential, a pK(a) of 7.2 has been determined for the GOx flavohydroquinone. At pH values ≤6.0, a dependency of the reduction mechanism and the GOx redox potential on the presence of halides, especially on Cl(-), was observed. For the development of glucose biosensors and glucose biofuel cell anodes working at physiological or neutral pH, the GOx redox potentials at pH 7.4 and pH 7.0 are of main interest. Here values of E(1/2 pH 7.4) = -97 ± 3 mV and E(1/2 pH 7.0) = -80 ± 4 mV have been determined.