We prepared Fe3O4 magnetic nanoparticles (MNPs) with the size of 10 nm by chemical coprecipitation. The effects of six aqueous-organic solvents, including tetrahydrofuran, dioxane, acetone, N, N-Dimethylformamide, methylalcohol, and Dimethyl Sulfoxide, on peroxidase mimetic activity of Fe3O4 MNPs were studied and compared with that of horseradish peroxidase (HRP). The relative activity of Fe3O4 MNPs droped sharply as the elevation of organic solvent concentration increased from 30% to 75% (V/V). In 15% organic solutions, the optimum activity of Fe3O4 MNPs was observed around 50oC, under pH 3.6. After being treated at different temperatures and pH in 15% organic solutions, even under 75% concentration, Fe3O4 MNPs still preserved most of the activity when reacting in aqueous phase. The catalytic performances of Fe3O4 MNPs under given conditions were generally more superior to that of HRP. For it costs lower and it is easy to be prepared and segregated magnetically for recycle, to use the magnetic nanoparticles as a substitution for HRP has potential to be applied into organic catalysis.