Myoglobin (Mb) was chemically modified with activated poly(ethylene oxide) (PEO) (average molecular weight of 2000) to solubilize it in various organic solvents. UV-vis, circular dichroism, and Raman spectroscopy were used to characterize the structure correlated with the electron-transfer reactions of PEO-modified Mb (PEO-Mb). Spectroscopic data indicated changes in heme coordination geometry for PEO-Mb in various organic solvents that are different from that in water. The Raman spectrum showed the characteristics of PEO-Mb in PEO oligomer (average MW of 200) in the five-coordinate high-spin state, which facilitates fast electron-transfer reactions between protein and the glassy carbon electrode. These results suggest heme environment effects on the properties of proteins in organic solvents.