This paper describes an electrochemically mediated enzyme reaction of polyethyleneglycol (PEG)-modified galactose oxidase (GAG) in organic solvents as well as in an aqueous solution. Catalytic currents were investigated in the presence of ferrocene derivatives as mediators and PEG-modified GAO in several organic solvents. The catalytic current due to the mediated enzyme reaction was obtained in acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide and dimethylsulfoxide (DMSO). Stability tests of PEG-modified GAO in organic solvents demonstrated that the initial I-k/I-d value was highest in acetonitrile; however, it gradually decreased. The PEG-modified GAO was more stable in DMSO. Reactivities of several mediators were investigated. Although a positively charged mediator indicated high reactivity in the aqueous solution, non-charged mediators such as ferrocene dimethanol and n-butyl ferrocene showed the highest activity in organic solvents. Substrate specificity demonstrated that the catalytic activity for benzyl alcohol in acetonitrile was greater than in aqueous solution. The effect of water content in acetonitrile was investigated. The catalytic activity decreased with the increase in water content.