The redox chemistry of several substituted benzoquinones was investigated by cyclic voltammetry at a glassy carbon electrode and candidates for inclusion in a mediated biosensor for use in flow analysis were selected on the basis of oxidation potential, electrochemical reversibility and solubility. Glucose sensors constructed by sequential deposition onto a carbon pellet of 2,6-dimethyl-1,4-benzoquinone, 2,3,5,6-tetramethyl-1,4-benzoquinone or phenyl-1,4-benzoquinone mediator solution, followed by glucose oxidase in polyvinylalcohol-Nafion solution, were tested for response to glucose using flow injection analysis. Sensors prepared from 2,6-dimethyl-1,4-benzoquinone gave highest sensitivity, with a linear range of response to glucose of 2.5 ± 40 mM. The use of an enzyme-free comparative electrode to eliminate the response from interferents was investigated.